RU2841244C2 - Chimeric antigen receptors and ways of use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to a T-cell or NK-cell, which includes a CAR molecule, which contains a first CAR that binds to BCMA (BCMA CAR), a second CAR that binds to CD19 (CD19 CAR), to a composition containing it, as well as to an isolated nucleic acid molecule coding a chimeric antigen receptor (CAR) molecule, where said nucleic acid molecule comprises a first nucleic acid sequence coding a first CAR that binds to BCMA (BCMA CAR), a second nucleic acid sequence coding a second CAR which binds to CD19 (CD19 CAR), and an isolated CAR molecule which binds to BCMA and CD19. Also disclosed is a vector, a cell containing said nucleic acid molecule, as well as a method of producing said cell. Invention also relates to a population of T-cells or NK-cells for expression of the CAR molecule.

EFFECT: invention is effective for providing anti-tumour immunity in a subject.

22 cl, 33 dwg, 34 tbl, 12 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application Ser. No. 62/940,509, filed November 26, 2019, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LIST

This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on November 24, 2020 is named N2067-7166WO_SL.txt and is 598,194 bytes in size.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates generally to immune effector cells (e.g., T cells or NK cells) engineered to express a chimeric antigen receptor (CAR), as well as compositions thereof and routes of use thereof.

BACKGROUND OF THE INVENTION

Adoptive cell transfer (ACT) therapy using T cells, particularly chimeric antigen receptor (CAR)-transduced T cells, has shown promise in several studies in hematological cancers. There is a need for methods and processes to improve the production of CAR-expressing cell-based therapeutic product, enhance product quality, and maximize the therapeutic efficacy of the product.

ESSENCE OF THE INVENTION

In one aspect, the present invention features a cell, e.g., an immune cell, e.g., a T cell or an NK cell, comprising a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain is a BCMA-binding domain. In some embodiments, the BCMA-binding domain comprises a BCMA-binding sequence disclosed herein, e.g., a CDR, VH, VL, or scFv sequence disclosed in Tables 3-15, 19, 20, 22, 26, and 31. In some embodiments, the second antigen-binding domain is a CD19-binding domain. In some embodiments, the CD19-binding domain comprises a CD19-binding sequence disclosed herein, e.g., a CDR, VH, VL, or scFv sequence disclosed in Tables 2, 19, 22, and 31.

In some embodiments, the present invention provides a cell comprising (a) a first antigen-binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC), and a heavy chain complementarity determining region 3 (CDR3 HC), and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 3 (CDR3 LC), wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of (i) SEQ ID NOs: 86, 130, 88, 95, 131, and 132, respectively; (ii) SEQ ID NOs: 44, 45, 84, 54, 55, and 56, respectively; or (iii) SEQ ID NOs: 179, 180, 181, 147, 182, and 183, respectively; and (b) a second antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are located in two chimeric antigen receptors (CARs). In some embodiments, the first antigen-binding domain and the second antigen-binding domain are located in a single CAR.

In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 86, 130, 88, 95, 131, and 132, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 86, 109, 88, 95, 114, and 115, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 86, 109, 88, 95, 114, and 97, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 93 or 112, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH is encoded by a nucleic acid sequence of SEQ ID NO: 260, 94, or 113, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 102, 118, or 124, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL is encoded by a nucleic acid sequence of SEQ ID NO: 261, 103, 119, or 125, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 93 and 102, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 112 and 118, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 112 and 124, respectively. In some embodiments, the first antigen-binding domain comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 105, 120, or 126, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 253, 106, 121, or 127, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is located in a first CAR. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 107, 226, 122, or 128, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 259, 258, 108, 123, or 129, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 84, 54, 55, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 68, 54, 55, and 56, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NOs: 78, 52, or 70, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH is encoded by the nucleic acid sequence of SEQ ID NOs: 79, 53, or 71, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL is encoded by the nucleic acid sequence of SEQ ID NO: 62 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 78 and 61, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 52 and 61, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 70 and 61, respectively. In some embodiments, the first antigen-binding domain comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 80, 64, or 72, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 81, 65, or 73, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is located in a first CAR. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 224, 82, 66, or 74, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 83, 67, or 75, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 179, 180, 181, 147, 182, and 183, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 137, 138, 139, 147, 148, and 149, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 160, 161, 162, 147, 170, and 171, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 145 or 168, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH is encoded by a nucleic acid sequence of SEQ ID NO: 146 or 169, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 154 or 173, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the VL is encoded by the nucleic acid sequence of SEQ ID NO: 155 or 174, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NO: 145 and 154, respectively. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NO: 168 and 173, respectively. In some embodiments, the first antigen-binding domain comprises a single-chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 156 or 175, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 157 or 176, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is located in a first CAR. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 158 or 177, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 159 or 178, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, provided herein is a cell comprising (a) a first antigen-binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises (i) a VH comprising the HC CDR1, HC CDR2, and HC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, and a VL comprising the LC CDR1, LC CDR2, and LC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243; (ii) a VH and VL comprising the amino acid sequences of SEQ ID NO: 239 and 242, respectively, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243; or (iii) an scFv comprising the amino acid sequence of SEQ ID NO: 200; and (b) a second antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are located in two chimeric antigen receptors (CARs). In some embodiments, the first antigen-binding domain and the second antigen-binding domain are located in a single CAR. In some embodiments, the second antigen-binding domain binds to an antigen selected from CD19, CD5, CD10, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, FcRn5, FcRn2, CS-1, CXCR4, 5, 7, IL-7/3R, IL7/4/3R, or IL4R, wherein the B cell antigen is optionally selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD123, CD33, CD34, CLL-1, folate receptor beta, or FLT3. In some embodiments, the second antigen-binding domain binds to CD19. In some embodiments, the second antigen-binding domain binds to an antigen selected from EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (e.g., ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, receptor folate beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, hsp 70-2 mutant, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4 or a peptide of any of these antigens presented on MHC.

In some embodiments, the second antigen binding domain binds to CD19. In some embodiments, the second antigen binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and/or CDR3 LC of a CD19 binding sequence set forth in Table 19 or Table 22, such as CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively. In some embodiments, the second antigen-binding domain comprises a VH and/or VL of a CD19 binding sequence shown in Table 19 or Table 22, such as a VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the second antigen-binding domain comprises an scFv of a CD19 binding sequence shown in Table 19 or Table 22, such as an scFv comprising the amino acid sequence of SEQ ID NO: 211 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the second antigen-binding domain is located in a second CAR, wherein the CAR comprises a CAR with a CD19 binding sequence shown in Table 19 or Table 22, such as a CAR comprising the amino acid sequence of SEQ ID NO: 225 or 229 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the first antigen-binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR LC comprising the amino acid sequences of (a) SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively; (b) SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively; or (c) SEQ ID NOs: 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, the second antigen-binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively. In some embodiments, the first antigen-binding domain comprises a VH and VL comprising the amino acid sequences of (a) SEQ ID NO: 93 and 102, respectively; (b) SEQ ID NO: 78 and 61, respectively; or (c) SEQ ID NO: 52 and 61, respectively. In some embodiments, the second antigen-binding domain comprises a VH and VL comprising the amino acid sequences of SEQ ID NO: 250 and 251, respectively. In some embodiments, the first antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 105, 80, or 64. In some embodiments, the second antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 211. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 253, 106, 81, or 65. In some embodiments, the second antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 212.

In some embodiments, the first antigen-binding domain is located in a first CAR and the second antigen-binding domain is located in a second CAR. In some embodiments, the first CAR further comprises a first transmembrane domain and a first intracellular signaling domain. In some embodiments, the second CAR further comprises a second transmembrane domain and a second intracellular signaling domain.

In some embodiments, the first CAR is encoded by a first nucleic acid sequence and the second CAR is encoded by a second nucleic acid sequence, wherein the first and second nucleic acid sequences are located on separate nucleic acid molecules.

In some embodiments, a first CAR is encoded by a first nucleic acid sequence and a second CAR is encoded by a second nucleic acid sequence, wherein the first and second nucleic acid sequences are located on a single nucleic acid molecule. In some embodiments, a single nucleic acid molecule has the following configuration in a 5'-3' orientation: a nucleic acid sequence encoding a first antigen-binding domain - a nucleic acid sequence encoding a first transmembrane domain - a nucleic acid sequence encoding a first intracellular signaling domain - a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a second antigen-binding domain - a nucleic acid sequence encoding a second transmembrane domain - a nucleic acid sequence encoding a second intracellular signaling domain. In some embodiments, the individual nucleic acid molecule has the following configuration in a 5'-3' orientation: a nucleic acid sequence encoding a second antigen-binding domain - a nucleic acid sequence encoding a second transmembrane domain - a nucleic acid sequence encoding a second intracellular signaling domain - a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a first antigen-binding domain - a nucleic acid sequence encoding a first transmembrane domain - a nucleic acid sequence encoding a first intracellular signaling domain. In some embodiments, the linker comprises a self-cleavage site. In some embodiments, the linker comprises a P2A site, a T2A site, an E2A site, or an F2A site. In some embodiments, the linker comprises a P2A site. In some embodiments, the linker is encoded by the nucleic acid sequence of SEQ ID NO: 209 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the individual nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 215, 217, 219, 221, or 223 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, a single nucleic acid molecule encodes an amino acid sequence of SEQ ID NO: 214, 216, 218, 220, or 222, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the first antigen-binding domain and the second antigen-binding domain are located in a single CAR, wherein the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some embodiments, the first antigen-binding domain comprises a first VH (VH1) and a first VL (VL1), and the second antigen-binding domain comprises a second VH (VH2) and a second VL (VL2). In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH2 - optionally Linker 1 ("L1") - VL1 - optionally Linker 2 ("L2") - VH1 - optionally Linker 3 ("L3") - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VL1. In some embodiments VH1 and VL1 comprise the amino acid sequences of SEQ ID NO: 93 and 102, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments VH1 and VL1 comprise the amino acid sequences of SEQ ID NO: 333 and 334, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments VH1 and VL1 comprise the amino acid sequences of SEQ ID NO: 78 and 61, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments VH1 and VL1 comprise the amino acid sequences of SEQ ID NOs: 335 and 336, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH2 and VL2 comprise the amino acid sequences of SEQ ID NOs: 250 and 251, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH2 and VL2 comprise the amino acid sequences of SEQ ID NOs: 331 and 332, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, L1 or L3 comprises the amino acid sequence of SEQ ID NO: 5 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, L2 comprises the amino acid sequence of SEQ ID NO: 63 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 321-330, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 339-348, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, CAR is encoded by a nucleic acid molecule having the following configuration in 5'-3' orientation: a nucleic acid sequence encoding a first antigen-binding domain - optionally a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a second antigen-binding domain - a nucleic acid sequence encoding a transmembrane domain - a nucleic acid sequence encoding an intracellular signaling domain. In some embodiments CAR is encoded by a nucleic acid molecule having the following configuration in 5'-3' orientation: a nucleic acid sequence encoding a second antigen-binding domain - optionally a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a first antigen-binding domain - a nucleic acid sequence encoding a transmembrane domain - a nucleic acid sequence encoding an intracellular signaling domain.

In some embodiments, the CAR has the following configuration in N- to C-terminal orientation: a first antigen-binding domain - optionally a linker - a second antigen-binding domain - a transmembrane domain - an intracellular signaling domain. In some embodiments, the CAR has the following configuration in N- to C-terminal orientation: second antigen-binding domain - optionally a linker - first antigen-binding domain - transmembrane domain - intracellular signaling domain.

In some embodiments, the first antigen-binding domain or the second antigen-binding domain comprises VH and VL. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 5, 63, 104, or 243, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the transmembrane domain, the first transmembrane domain, or the second transmembrane domain comprises a transmembrane domain of a protein selected from the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain, the first transmembrane domain, or the second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the transmembrane domain, the first transmembrane domain or the second transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

In some embodiments, the first antigen-binding domain or the second antigen-binding domain is connected to the transmembrane domain, the first transmembrane domain, or the second transmembrane domain by a hinge region (e.g., a first or second hinge region). In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region is encoded by a nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region and the transmembrane domain comprise the amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region and the transmembrane domain are encoded by a nucleic acid sequence of SEQ ID NO: 203 or 213 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises a primary signaling domain (e.g., a first or second primary signaling domain). In some embodiments the primary signaling domain provides a functional signaling domain derived from CD3zeta, TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d. In some embodiments, the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the primary signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 20, 21, or 205, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises a costimulatory signaling domain (e.g., a first or second costimulatory signaling domain). In some embodiments, the costimulatory signaling domain provides a functional signaling domain derived from a class I MHC molecule, a TNF protein receptor, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an NK cell activating receptor, BTLA, a Toll-like receptor ligand, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD28-OX40, CD28-4-1BB, or a ligand that specifically binds to CD83. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 18 or 204 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3zeta. In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

In some embodiments, the CAR, the first CAR, or the second CAR further comprises a leader sequence (e.g., a first or second leader sequence). In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the leader sequence is encoded by the nucleic acid sequence of SEQ ID NO: 199 or 210 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the first leader sequence and the second leader sequence are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the first hinge region and the second hinge region are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the first primary signaling domain and the second primary signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the first leader sequence and the second leader sequence provide the same amino acid sequences (e.g., the first leader sequence and the second leader sequence provide the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first leader sequence and the second leader sequence provide different amino acid sequences. In some embodiments, the first hinge region and the second hinge region comprise the same amino acid sequences (e.g., the first hinge region and the second hinge region comprise the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first hinge region and the second hinge region comprise different amino acid sequences. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequences (e.g., the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first transmembrane domain and the second transmembrane domain comprise different amino acid sequences. In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequences. In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain comprise different amino acid sequences. In some embodiments, the first primary signaling domain and the second primary signaling domain comprise the same amino acid sequence (e.g., the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first primary signaling domain and the second primary signaling domain comprise different amino acid sequences. In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise the same amino acid sequence (e.g., the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise different amino acid sequences (e.g., the first and second costimulatory signaling domains comprise a 4-1BB costimulatory domain sequence and a CD28 costimulatory domain sequence, respectively, or comprise a CD28 costimulatory domain sequence and a 4-1BB costimulatory domain sequence, respectively). In some embodiments, the first leader sequence and the second leader sequence are encoded by nucleic acid sequences comprising SEQ ID NOs: 199 and 210, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first leader sequence and the second leader sequence are encoded by nucleic acid sequences comprising SEQ ID NOs: 210 and 199, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first hinge region and the second hinge region are encoded by nucleic acid sequences comprising SEQ ID NOs: 337 and 13, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first hinge region and the second hinge region are encoded by nucleic acid sequences comprising SEQ ID NO: 13 and 337, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by nucleic acid sequences comprising SEQ ID NO: 338 and 17, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 17 and 338, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 204 and 18, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by nucleic acid sequences of SEQ ID NOs: 18 and 204, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first primary signaling domain and the second primary signaling domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 205 and 21, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first primary signaling domain and the second primary signaling domain are encoded by nucleic acid sequences comprising SEQ ID NO: 21 and 205, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the CAR, the first CAR or the second CAR is encoded by a nucleic acid molecule comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In some embodiments, provided herein is a nucleic acid molecule comprising (a) a first nucleic acid sequence encoding a first antigen-binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC), and a heavy chain complementarity determining region 3 (CDR3 HC), and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 3 (CDR3 LC), wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC comprise the amino acid sequences of (i) SEQ ID NO: 86, 130, 88, 95, 131 and 132, respectively; (ii) SEQ ID NO: 44, 45, 84, 54, 55 and 56, respectively, or (iii) SEQ ID NO: 179, 180, 181, 147, 182 and 183, respectively; and (b) a second nucleic acid sequence encoding a second antigen-binding domain.

In some embodiments, the isolated nucleic acid molecule comprises a first nucleic acid molecule and a second nucleic acid molecule that are separate nucleic acid molecules, and wherein the first nucleic acid sequence is located in the first nucleic acid molecule and the second nucleic acid sequence is located in the second nucleic acid molecule.

In some embodiments, provided herein is a nucleic acid molecule comprising (a) a first nucleic acid sequence encoding a first antigen-binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises (i) a VH comprising HC CDR1, HC CDR2, and HC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, and a VL comprising LC CDR1, LC CDR2, and LC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243; (ii) VH and VL comprising the amino acid sequences of SEQ ID NO: 239 and 242, respectively, wherein VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243; or (iii) an scFv comprising the amino acid sequence of SEQ ID NO: 200; and (b) a second nucleic acid sequence encoding a second antigen-binding domain.

In some embodiments, provided herein is a nucleic acid molecule comprising a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first CAR comprises a first antigen-binding domain that is a BCMA binding domain, a first transmembrane domain, and a first intracellular signaling domain, and wherein the second CAR comprises a second antigen-binding domain that is a CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain, wherein (i) the first antigen-binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR LC comprising the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively, and the second antigen-binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR LC comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively; (ii) the first antigen-binding domain comprises a VH and VL comprising the amino acid sequences of SEQ ID NOs: 93 and 102, respectively, and the second antigen-binding domain comprises a VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively; (iii) the first antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 105, and the second antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 211; (iv) the first CAR comprises the amino acid sequence of SEQ ID NO: 107 or 226, and the second CAR comprises the amino acid sequence of SEQ ID NO: 225 or 229; or (v) the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 271.

In some embodiments, provided herein is a nucleic acid molecule comprising a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first CAR comprises a first antigen-binding domain that is a BCMA binding domain, a first transmembrane domain, and a first intracellular signaling domain, and wherein the second CAR comprises a second antigen-binding domain that is a CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain, wherein (i) the first antigen-binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR LC comprising the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively, the second antigen-binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively; (ii) the first antigen-binding domain comprises a VH and VL comprising the amino acid sequences of SEQ ID NOs: 78 and 61, respectively, and the second antigen-binding domain comprises a VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively; (iii) the first antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 80, and the second antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 211; (iv) the first CAR comprises the amino acid sequence of SEQ ID NO: 82 or 224, and the second CAR comprises the amino acid sequence of SEQ ID NO: 225 or 229; or (v) the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 215.

In some embodiments, provided herein is a polypeptide molecule encoded by a nucleic acid molecule disclosed herein.

In some embodiments, provided herein is a CAR, wherein the CAR comprises (a) a first antigen-binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC), and a heavy chain complementarity determining region 3 (CDR3 HC), and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 3 (CDR3 LC), wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of (i) SEQ ID NOs: 86, 130, 88, 95, 131 and 132, respectively; (ii) SEQ ID NOs: 44, 45, 84, 54, 55 and 56, respectively, or (iii) SEQ ID NOs: 179, 180, 181, 147, 182 and 183, respectively; and (b) a second antigen-binding domain.

In some embodiments, provided herein is a CAR, wherein the CAR comprises (a) a first antigen binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises (i) a VH comprising the HC CDR1, HC CDR2, and HC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, and a VL comprising the LC CDR1, LC CDR2, and LC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243; (ii) a VH and VL comprising the amino acid sequences of SEQ ID NO: 239 and 242, respectively, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243; or (iii) an scFv comprising the amino acid sequence of SEQ ID NO: 200; and (b) a second antigen-binding domain.

In some embodiments, provided herein is a vector comprising a nucleic acid molecule disclosed herein or a nucleic acid molecule encoding a CAR disclosed herein. In some embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the vector comprises an EF-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 11.

In some embodiments, provided herein is a cell comprising a nucleic acid molecule disclosed herein, a nucleic acid molecule encoding a CAR disclosed herein, a polypeptide disclosed herein, a CAR disclosed herein, or a vector disclosed herein. In some embodiments, the cell is a T cell or an NK cell.

In some embodiments, disclosed herein is a method of producing a cell comprising transducing the cell with a vector disclosed herein, wherein the cell is optionally a T cell or an NK cell. In some embodiments, disclosed herein is a method of producing an RNA engineered cell comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid molecule disclosed herein, a nucleic acid molecule encoding a CAR disclosed herein. In some embodiments, the cell is a T cell or an NK cell.

In some embodiments, disclosed herein is a method for producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising (i) contacting the population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) with (e.g., binding) an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule as disclosed herein or a nucleic acid molecule encoding a CAR as disclosed herein, thereby producing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (iii) collecting the population of cells (e.g., T cells) for storage (e.g., reconstituting the population of cells in a cryopreservation medium) or administration, wherein:

(a) step (ii) is carried out together with step (i) or not later than 20 hours after the start of step (i), such as not later than 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), such as not later than 18 hours after the start of step (i), and

step (iii) is carried out no later than 30 (e.g. 26) hours after the start of step (i), such as no later than 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (i), such as no later than 24 hours after the start of step (i),

(b) step (ii) is carried out together with step (i) or not later than 20 hours after the start of step (i), such as not later than 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), such as not later than 18 hours after the start of step (i), and

step (iii) is carried out no later than 30 hours after the start of step (ii), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii), or

(c) the population of cells from step (iii) is not expanded or is expanded by an increase in number of no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of living cells, compared to the population of cells at the start of step (i),

wherein the nucleic acid molecule in step (ii) is optionally in a viral vector, wherein the nucleic acid molecule in step (ii) is optionally an RNA molecule in a viral vector, wherein step (ii) optionally comprises transducing a population of cells (e.g. T cells) with a viral vector containing a nucleic acid molecule encoding a CAR.

In some embodiments, disclosed herein is a method for producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising (1) contacting the population of cells (e.g., T cells, e.g., T cells isolated from a frozen leukapheresis product) with a cytokine selected from IL-2, IL-7, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-21, IL-6 (e.g., IL-6/sIL-6Ra), or a combination thereof, (2) contacting the population of cells (e.g., T cells) with a nucleic acid molecule as disclosed herein or a nucleic acid molecule encoding a CAR as disclosed herein, thereby producing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (3) collecting the population of cells (e.g., T cells) for storage (e.g. repopulation of cells in cryopreservation medium) or administration where:

(a) step (2) is carried out together with step (1) or not later than 5 hours after the start of step (1), for example not later than 1, 2, 3, 4 or 5 hours after the start of step (1), and

stage (3) is carried out no later than 26 hours after the start of stage (1), for example, no later than 22, 23 or 24 hours after the start of stage (1), for example, no later than 24 hours after the start of stage (1), or

(b) the population of cells from step (3) is not expanded or is expanded by an increase in number of no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of living cells, compared to the population of cells at the start of step (1),

wherein the nucleic acid molecule in step (2) is optionally in a viral vector, wherein the nucleic acid molecule in step (ii) is optionally an RNA molecule in a viral vector, wherein step (ii) optionally comprises transducing a population of cells (e.g. T cells) with a viral vector containing a nucleic acid molecule encoding a CAR.

In some embodiments, disclosed herein is a population of cells engineered to express a CAR ("CAR-expressing population of cells"), wherein the population comprises: (a) about the same percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, compared to the percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ cells, in the same population of cells prior to being engineered to express a CAR; (b) a change in the number of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, of from about 5% to about 10%, such as compared to the percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, in the same population of cells before they were engineered to express the CAR; (c) an increased percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, such as increased by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, compared to the percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, in the same population of cells prior to engineering them to express the CAR; (d) approximately the same percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (e) a change in the number of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, of from about 5% to about 10% compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (f) a reduced percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, such as reduced by at least 20, 25, 30, 35, 40, 45 or 50%, compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (g) approximately the same percentage of memory T stem cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of memory T stem cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same cell population before they were engineered to express the CAR; (h) a change in the number of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, of between about 5% and about 10%, compared to the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells prior to their engineering to express the CAR; or (i) an increased percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells before they were engineered to express a CAR. In some embodiments, the population comprises a cell as disclosed herein. In some embodiments, the population comprises a cell comprising a dual CAR or a diabody format CAR as disclosed herein. In some embodiments, the population comprises a cell comprising (a) a first antigen-binding domain that is a BCMA binding domain, wherein the BCMA binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC), and a heavy chain complementarity determining region 3 (CDR3 HC), and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 3 (CDR3 LC), wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of (i) SEQ ID NO: 1 ID NO: 86, 130, 88, 95, 131 and 132, respectively; (ii) SEQ ID NO: 44, 45, 84, 54, 55 and 56, respectively, or (iii) SEQ ID NO: 179, 180, 181, 147, 182 and 183, respectively; and (b) a second antigen-binding domain.

In some embodiments, disclosed herein is a pharmaceutical composition comprising a cell disclosed herein or a population of cells disclosed herein and a pharmaceutically acceptable carrier.

In some embodiments, the population of cells is obtained by a method disclosed herein. In some embodiments, the population comprises:

(a) the first population of cells containing CAR for BCMA but not CAR for CD19;

(b) a second population of cells containing a CAR for CD19 but not a CAR for BCMA; and

(c) a third population of cells containing both CAR for BCMA and CAR for CD19.

In some embodiments:

(i) the total number of viable cells in the combined second and third populations is less than or equal to approximately 110% (e.g., less than or equal to approximately 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations;

(ii) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations; and/or

(iii) the total number of viable cells in the combined first and third populations is greater than or equal to approximately 5% (e.g., greater than or equal to approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the population.

In some embodiments, the population further comprises a fourth population of cells that do not contain CAR.

In some embodiments:

(i) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations;

(ii) the total number of viable cells in the second population is less than or equal to from about 45% to about 50% (e.g., about 47%); from about 50% to about 55% (e.g., about 53%); from about 60% to about 65% (e.g., about 63%); or from about 80% to about 85% (e.g., about 82%) of the total number of viable cells in the combined first and third populations.

In some embodiments, disclosed herein is a method of providing anti-tumor immunity in a subject comprising administering to the subject an effective amount of a cell disclosed herein, a population of cells disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, disclosed herein is a method of treating a subject having a disease associated with BCMA expression comprising administering to the subject an effective amount of a cell disclosed herein, a population of cells disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the disease associated with BCMA expression is (i) cancer or a malignancy or a precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome, or preleukemia, or (ii) a non-cancer indication associated with BCMA expression. In some embodiments, the disease is a hematological cancer or a solid cancer. In some embodiments, the disease is selected from acute leukemia, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorders (e.g., asymptomatic myeloma ("smoldering" multiple myeloma or smoldering myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloidosis in the form of light chain amyloidosis or POEMS syndrome (also known as POEMS syndrome) Crowe-Fukasa, Takatsuki disease, and PEP syndrome)) or combinations thereof. In some embodiments, the disease is multiple myeloma.

In some embodiments, the population of cells or pharmaceutical composition is administered to the subject at a dose of from about 1×10 ⁶ to about 1×10 ⁸ (e.g., from about 2×10 ⁶ to about 5×10 ⁷ , from about 5×10 ⁶ to about 2×10 ⁷ , from about 1×10 ⁶ to about 1×10 ⁷ , from about 1×10 ⁷ to about 1×10 ⁸ , from about 1×10 ⁶ to about 3×10 ⁶ , from about 2×10 ⁶ to about 4×10 ⁶ , from about 3×10 ⁶ to about 5×10 ⁶ , from about 4×10 ⁶ to about 6×10 ⁶ , from about 5×10 ⁶ to about 7×10 ⁶ , from about 6×10 ⁶ to about 8×10 ⁶ , from about 7×10 ⁶ to about 9×10 ⁶ , from about 8×10 ⁶ to about 1×10 ⁷ , from about 9×10 ⁶ to about 2×10 ⁷ , from about 1×10 ⁷ to about 3×10 ⁷ , from about 2×10 ⁷ to about 4×10 ⁷ , from about 3×10 ⁷ to about 5×10 ⁷ , from about 4×10 ⁷ to about 6×10 ⁷ , from about 5×10 ⁷ to about 7×10 ⁷ , from about 6×10 ⁷ to about 8×10 ⁷ , from about 7×10 ⁷ to about 9×10 ⁷ , from about 8×10 ⁷ to about 1×10 ⁸ , about 1×10 ⁶ , about 2×10 ⁶ , about 3×10 ⁶ , about 4×10 ⁶ , about 5×10 ⁶ , about 6×10 ⁶ , about 7×10 ⁶ , about 8×10 ⁶ , about 9×10 ⁶ , about 1×10 ⁷ , about 2×10 ⁷ , about 3×10 ⁷ , about 4×10 ⁷ , about 5×10 ⁷ , about 6×10 ⁷ , about 7×10 ⁷ , about 8×10 ⁷ , about 9×10 ⁷ or about 1×10 ⁸ ) CAR-positive viable cells (e.g., BCMA-CAR+ T cells). In some embodiments, the population of cells or pharmaceutical composition is administered to the subject at a dose of from about 5×10 ⁶ to about 2×10 ⁷ CAR-positive viable cells (e.g., BCMA-CAR+ T cells).

In some embodiments, the population of cells or the pharmaceutical composition is administered to the subject in one or more (e.g., 2, 3, 4, or more) doses. In some embodiments, the population of cells or the pharmaceutical composition is administered to the subject in two doses. In some embodiments, the one or more doses comprise a first dose and a second dose, wherein the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the first dose is greater than, equal to, or less than the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the second dose.

In some embodiments, the one or more doses comprise a first dose and a second dose, wherein:

(a) the first dose comprises from about 1×10 ⁶ to about 1×10 ⁷ (e.g., from about 2×10 ⁶ to about 8×10 ⁶ , from about 4×10 ⁶ to about 6×10 ⁶ , from about 1×10 ⁶ to about 5×10 ⁶ , from about 5×10 ⁶ to about 1×10 ⁷ , from about 1×10 ⁶ to about 3×10 ⁶ , from about 2×10 ⁶ to about 4×10 ⁶ , from about 3×10 ⁶ to about 5×10 ⁶ , from about 4×10 ⁶ to about 6×10 ⁶ , from about 5×10 ⁶ to about 7×10 ⁶ , from about 6×10 ⁶ to about 8×10 ⁶ , from about 7×10 ⁶ to about 9×10 ⁶ , from about 8×10 ⁶ to about 1×10 ⁷ , about 1×10 ⁶ , about 2×10 ⁶ , about 3×10 ⁶ , about 4×10 ⁶ , about 5×10 ⁶ , about 6×10 ⁶ , about 7×10 ⁶ , about 8×10 ⁶ , about 9×10 ⁶ , or about 1×10 ⁷ ) of viable CAR-positive cells (e.g., BCMA-CAR+ T cells);

(b) the second dose comprises from about 1×10 ⁷ to about 1×10 ⁸ (e.g., from about 2×10 ⁷ to about 8×10 ⁷ , from about 4×10 ⁷ to about 6×10 ⁷ , from about 1×10 ⁷ to about 5×10 ⁷ , from about 5×10 ⁷ to about 1×10 ⁸ , from about 1×10 ⁷ to about 3×10 ⁷ , from about 2×10 ⁷ to about 4×10 ⁷ , from about 3×10 ⁷ to about 5×10 ⁷ , from about 4×10 ⁷ to about 6×10 ⁷ , from about 5×10 ⁷ to about 7×10 ⁷ , from about 6×10 ⁷ to about 8×10 ⁷ , from about 7×10 ⁷ to about 9×10 ⁷ , from about 8×10 ⁷ to about 1×10 ⁸ , about 1×10 ⁷ , about 2×10 ⁷ , about 3×10 ⁷ , about 4×10 ⁷ , about 5×10 ⁷ , about 6×10 ⁷ , about 7×10 ⁷ , about 8×10 ⁷ , about 9×10 ⁷ or about 1×10 ⁸ ) CAR-positive viable cells (e.g., BCMA-CAR+ T cells);

(c) the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the first dose is no more than 1/X, where X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, of the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the second dose; and/or

(d) the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the first dose is from about 1% to 100% (e.g., from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 10% to about 50%, from about 50% to about 90%, from about 10% to about 30%, from about 20% to about 40%, from about 30% to about 50%, from about 50% to about 70%, from about 60% to about 80%, or from about 70% to about 90%) of the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the second dose.

In some embodiments, the first dose comprises about 5×10 ⁶ viable CAR-positive cells (e.g., BCMA-CAR+ T cells). In some embodiments, the second dose comprises about 1×10 ⁷ or about 2×10 ⁷ viable CAR-positive cells (e.g., BCMA-CAR+ T cells).

In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from (i) a PD-1 inhibitor, wherein the PD-1 inhibitor is optionally selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224; (ii) a PD-L1 inhibitor, wherein the PD-L1 inhibitor is optionally selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559; (iii) a LAG-3 inhibitor, wherein the LAG-3 inhibitor is optionally selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280 and REGN3767; (iv) a TIM-3 inhibitor, wherein the TIM-3 inhibitor is optionally selected from the group consisting of MBG453, TSR-022 and LY3321367; (v) a CTLA-4 inhibitor, wherein the CTLA-4 inhibitor is optionally ipilimumab or tremelimumab; (vi) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha subunit (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide, such as hetIL-15; (vii) an interleukin-12 (IL-12) polypeptide; or (viii) an mTOR inhibitor, wherein the mTOR inhibitor is optionally RAD001 or rapamycin.

In some embodiments, provided herein is a cell comprising (a) a first CAR comprising a first antigen-binding domain that binds to a first antigen, a first transmembrane domain, and a first intracellular signaling domain (e.g., a first primary signaling domain and/or a first costimulatory signaling domain), wherein the first CAR optionally further comprises a first leader sequence and/or a first hinge region; and (b) a second CAR comprising a second antigen-binding domain that binds a second antigen, a second transmembrane domain, and a second intracellular signaling domain (e.g., a second primary signaling domain and/or a second costimulatory signaling domain), wherein the second CAR optionally further comprises a second leader sequence and/or a second hinge region, wherein (i) the first leader sequence and the second leader sequence are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first and second leader sequences optionally comprise the same amino acid sequences; (ii) the first hinge region and the second hinge region are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first and second hinge regions optionally comprise the same amino acid sequences; (iii) the first transmembrane domain and the second transmembrane domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first and second transmembrane domains optionally comprise the same amino acid sequences; and/or (iv) the first intracellular signaling domain and the second intracellular signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein optionally the first primary signaling domain and the second primary signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%) and/or the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%).

In some embodiments, provided herein is a nucleic acid molecule comprising (a) a first nucleic acid sequence encoding a first CAR, wherein the first CAR comprises a first antigen-binding domain that binds a first antigen, a first transmembrane domain, and a first intracellular signaling domain (e.g., a first primary signaling domain and/or a first costimulatory signaling domain), wherein the first CAR optionally further comprises a first leader sequence and/or a first hinge region; and (b) a second nucleic acid sequence encoding a second CAR, wherein the second CAR comprises a second antigen-binding domain that binds a second antigen, a second transmembrane domain, and a second intracellular signaling domain (e.g., a second primary signaling domain and/or a second costimulatory signaling domain), wherein the second CAR optionally further comprises a second leader sequence and/or a second hinge region, wherein (i) the first leader sequence and the second leader sequence are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first and second leader sequences optionally comprise the same amino acid sequences; (ii) the first hinge region and the second hinge region are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first and second hinge regions optionally comprise the same amino acid sequences; (iii) the first transmembrane domain and the second transmembrane domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first and second transmembrane domains optionally comprise the same amino acid sequences; and/or (iv) the first intracellular signaling domain and the second intracellular signaling domain are encoded by different nucleic acid sequences (e.g., differing by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%), wherein the first intracellular signaling domain and the second intracellular signaling domain optionally comprise the same amino acid sequences.

In some embodiments, the first and second leader sequences comprise the same amino acid sequences. Without being bound by any theory, it is believed that such a nucleic acid molecule exhibits less recombination than an otherwise similar nucleic acid molecule in which the first leader sequence and the second leader sequence are encoded by the same nucleic acid sequences.

In some embodiments, the first and second hinge regions comprise the same amino acid sequences. Without being limited by theory, it is believed that such a nucleic acid molecule exhibits less recombination than an otherwise similar nucleic acid molecule in which the first hinge region and the second hinge region are encoded by the same nucleic acid sequences.

In some embodiments, the first and second transmembrane domains comprise the same amino acid sequences. Without being limited by theory, it is believed that such a nucleic acid molecule exhibits less recombination than an otherwise similar nucleic acid molecule in which the first transmembrane domain and the second transmembrane domain are encoded by the same nucleic acid sequences.

In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequences. Without being limited by theory, it is believed that such a nucleic acid molecule exhibits less recombination than an otherwise similar nucleic acid molecule in which the first intracellular signaling domain and the second intracellular signaling domain are encoded by the same nucleic acid sequences.

In some embodiments, the first primary signaling domain and the second primary signaling domain comprise the same amino acid sequences. Without being limited by theory, it is believed that such a nucleic acid molecule exhibits less recombination than an otherwise similar nucleic acid molecule in which the first primary signaling domain and the second primary signaling domain are encoded by the same nucleic acid sequences.

In some embodiments, the first primary signaling domain and the second primary signaling domain comprise different amino acid sequences.

In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise the same amino acid sequences. Without being limited by theory, it is believed that such a nucleic acid molecule exhibits less recombination than an otherwise similar nucleic acid molecule in which the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by the same nucleic acid sequences.

In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise different amino acid sequences (e.g., the first and second costimulatory signaling domains comprise a 4-1BB costimulatory domain sequence and a CD28 costimulatory domain sequence, respectively, or comprise a CD28 costimulatory domain sequence and a 4-1BB costimulatory domain sequence, respectively).

In some embodiments, the first leader sequence and the second leader sequence comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first leader sequence and the second leader sequence are encoded by nucleic acid sequences comprising SEQ ID NO: 199 and 210, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) or SEQ ID NO: 210 and 199, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first hinge region and the second hinge region comprise the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first hinge region and the second hinge region are encoded by nucleic acid sequences comprising SEQ ID NOs: 337 and 13, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) or SEQ ID NOs: 13 and 337, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 338 and 17, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) or SEQ ID NOs: 17 and 338, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 204 and 18, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) or SEQ ID NOs: 18 and 204, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first primary signaling domain and the second primary signaling domain are encoded by nucleic acid sequences comprising SEQ ID NO: 205 and 21, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) or SEQ ID NO: 21 and 205, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first and second antigens are different. In some embodiments, the first or second antigen is selected from BCMA, CD19, CD5, CD10, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, FcRn5, FcRn2, CS-1, CXCR4, 5, 7, IL-7/3R, IL7/4/3R, or IL4R, wherein the B cell antigen is optionally selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD123, CD33, CD34, CLL-1, folate receptor beta, FLT3, EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (eg, ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, hsp 70-2 mutant, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC. In some embodiments, the first or second antigen-binding domain comprises a CDR, VH, VL, or scFv disclosed herein, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, provided herein is a CAR comprising a first VH (VH1), a first VL (VL1), a second VH (VH2), a second VL (VL2), a transmembrane domain, and an intracellular signaling domain, wherein VH1 and VL1 bind a first antigen and VH2 and VL2 bind a second antigen. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optionally Linker 1 ("L1") - VH2 - optionally Linker 2 ("L2") - VL2 - optionally Linker 3 ("L3") - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - Linker 1 ("L1") - VH2 - Linker 2 ("L2") - VL2 - Linker 3 ("L3") - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - L1 - VL2 - L2 - VH2 - L3 - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL1 - L1 - VH2 - L2 - VL2 - L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL1 - L1 - VL2 - L2 - VH2 - L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH2 - L1 - VH1 - L2 - VL1 - L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH2 - L1 - VL1 - L2 - VH1 - L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL2 - L1 - VH1 - L2 - VL1 - L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VL2 - L1 - VL1 - L2 - VH1 - L3 - VH2. In some embodiments, L1 or L3 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, L2 comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the CAR has the following configuration from N-terminus to C-terminus: (i) VH1 - optionally linker 1 ("L1") - VH2 - optionally linker 2 ("L2") - VL2 - optionally linker 3 ("L3") - VL1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (ii) VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (iii) VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (iv) VL1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VH1 - optional hinge region - transmembrane domain - intracellular signaling domain; (v) VH2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VL2 - optional hinge region - transmembrane domain - intracellular signaling domain; (vi) VH2 - optional L1 - VL1 - optional L2 - VH1 - optional L3 - VL2 - optional hinge region - transmembrane domain - intracellular signaling domain; (vii) VL2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VH2 - optional hinge region - transmembrane domain - intracellular signaling domain; or (viii) VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2 - optionally hinge region - transmembrane domain - intracellular signaling domain. In some embodiments, the first and second antigens are different. In some embodiments, the first or second antigen is selected from BCMA, CD19, CD5, CD10, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, FcRn5, FcRn2, CS-1, CXCR4, 5, 7, IL-7/3R, IL7/4/3R, or IL4R, wherein the B cell antigen is optionally selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD123, CD33, CD34, CLL-1, folate receptor beta, FLT3, EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (eg, ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, hsp 70-2 mutant, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC. In some embodiments, VH1, VL1, VH2, or VL2 comprises a CDR, VH, or VL sequence disclosed herein, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region, transmembrane domain, or intracellular signaling domain (e.g., primary signaling domain and/or costimulatory signaling domain) comprises a hinge region sequence, transmembrane domain sequence, or intracellular signaling domain sequence (e.g., primary signaling domain sequence and/or costimulatory signaling domain sequence) disclosed herein, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, provided herein is a nucleic acid molecule encoding a CAR in diabody format as disclosed herein. In some embodiments, provided herein is a vector comprising a nucleic acid molecule encoding a CAR in diabody format as disclosed herein. In some embodiments, provided herein is a cell comprising a CAR as disclosed herein, a nucleic acid molecule encoding a CAR in diabody format as disclosed herein, or a vector comprising a nucleic acid molecule encoding a CAR in diabody format as disclosed herein. In some embodiments, provided herein is a pharmaceutical composition that comprises a cell comprising a CAR in diabody format as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, provided herein is a method for producing a cell comprising a CAR in diabody format as disclosed herein. In some embodiments, disclosed herein is a method of treating a subject, such as a subject with cancer, using a cell comprising a CAR in a diabody format as disclosed herein.

In some embodiments, the present invention relates to methods for producing immune effector cells (e.g., T cells or NK cells) engineered to express CARs and compositions produced using such methods. The methods disclosed herein (e.g., the ARM method or the cytokine method disclosed herein) can be used to produce cells expressing the dual CARs or diabody format CARs disclosed herein. Also disclosed are methods for using such compositions to treat a disease, e.g., cancer, in a subject.

In some embodiments, the present invention provides a method of producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising: (i) contacting the population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) with (e.g., binding to) an agent that stimulates the CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a CAR, thereby producing a population of cells (e.g., T cells) containing the nucleic acid molecule, and (iii) collecting the population of cells (e.g., T cells) for storage (e.g., reconstituting the population of cells in a cryopreservation medium) or administration, wherein: (a) step (ii) is performed in conjunction with step (i) or no later than 20 hours after the start of step (i), such as no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), such as no later than 18 hours after the start of step (i), and step (iii) is performed no later than 26 hours after the start of step (i), such as no later than 22, 23, 24, or 25 hours after the start of step (i), for example, not later than 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and step (iii) is carried out not later than 30 hours after the start of step (ii), for example not later than 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not expanded or is expanded by an increase in number of no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, such as no more than 10%, such as as assessed by the number of live cells, compared to the population of cells at the beginning of step (i). In some embodiments, the nucleic acid molecule of step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is in a viral vector, such as a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (ii) is in a non-viral vector. In some embodiments, the nucleic acid molecule of step (ii) is in a plasmid. In some embodiments, the nucleic acid molecule of step (ii) is not in any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR. In some embodiments, step (ii) is performed together with step (i). In some embodiments, step (ii) is performed no later than 20 hours after the start of step (i). In some embodiments, step (ii) is performed no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i). In some embodiments, step (ii) is performed no later than 18 hours after the start of step (i). In some embodiments, step (iii) is performed no later than 26 hours after the start of step (i). In some embodiments, step (iii) is performed no later than 22, 23, 24, or 25 hours after the start of step (i). In some embodiments, step (iii) is performed no later than 24 hours after the start of step (i). In some embodiments, step (iii) is performed no later than 30 hours after the start of step (ii). In some embodiments, step (iii) is performed no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii). In some embodiments, the nucleic acid molecule encoding the CAR is a nucleic acid molecule as disclosed herein. In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR. In some embodiments, the first and second nucleic acid sequences are located on a single nucleic acid molecule, such as where the first nucleic acid sequence and the second nucleic acid sequence are separated by a third nucleic acid sequence encoding a self-cleavage site (e.g., a P2A site, a T2A site, an E2A site, or an F2A site). In some embodiments, the first and second nucleic acid sequences are located on separate nucleic acid molecules. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a CAR, wherein the CAR comprises a first VH (VH1), a first VL (VL1), a second VH (VH2), a second VL (VL2), a transmembrane domain, and an intracellular signaling domain, wherein VH1 and VL1 bind a first antigen and VH2 and VL2 bind a second antigen, wherein VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optional Linker 1 ("L1") - VH2 - optional Linker 2 ("L2") - VL2 - optional Linker 3 ("L3") - VL1, VH1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VL1, VL1 - optional L1 - VH2 - optional L2 - VL2 - optional L3 - VH1, VL1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VH1, VH2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VL2, VH2 - optional L1 - VL1 - optional L2 - VH1 - optional L3 - VL2, VL2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VH2 or VL2 - optional L1 - VL1 - optional L2 - VH1 - optional L3 - VH2.

In some embodiments, the cell population of step (iii) is not expanded. In some embodiments, the cell population of step (iii) is expanded by an increase in abundance of no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, such as as measured by the number of living cells, compared to the cell population at the beginning of step (i). In some embodiments, the cell population of step (iii) is expanded by an increase in abundance of no more than 10%, such as as measured by the number of living cells, compared to the cell population at the beginning of step (i).

In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first and second nucleic acid sequences are located on separate nucleic acid molecules.

In some embodiments, the first and second nucleic acid molecules are in separate viral vectors, and wherein step (ii) comprises transducing a population of cells (e.g., T cells) with a first viral vector comprising a nucleic acid molecule encoding a first CAR and a second viral vector comprising a second nucleic acid molecule encoding a second CAR.

In some embodiments, the first CAR comprises a BCMA binding domain (e.g., a BCMA CAR) and the second CAR comprises a CD19 binding domain (e.g., a CD19 CAR).

In some embodiments, in step (ii), the population of cells is contacted with the first viral vector at a multiplicity of infection (MOI) that is greater than, equal to, or less than the MOI at which the population of cells is contacted with the second viral vector. In some embodiments, in step (ii), the population of cells is contacted with the first viral vector at a multiplicity of infection (MOI) that is greater than the MOI at which the population of cells is contacted with the second viral vector.

In some embodiments, in step (ii), the population of cells is contacted with a first viral vector at a first MOI and with a second viral vector at a second MOI, such that the resulting population of cells comprises a first population of cells that contain a CAR for BCMA but not a CAR for CD19, a second population of cells that contain a CAR for CD19 but not a CAR for BCMA, and a third population of cells that contain both a CAR for BCMA and a CAR for CD19, where:

(a) the total number of viable cells in the combined second and third populations is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10;

(b) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations, e.g., when determined by the method described in Example 10;

(c) the total number of viable cells in the combined first and third populations is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the resulting population, e.g., when determined by the method described in Example 10;

(d) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10; or

(e) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000%, or more) of the total number of viable cells in the second population, e.g., as determined by the method described in Example 10. In some embodiments, step (ii) comprises contacting the population of cells with a second viral vector at an MOI, e.g., an MOI that is sufficiently lower than the MOI at which the population of cells is contacted with the first viral vector, such that the resulting population of cells:

(a) the total number of viable cells in the combined second and third populations is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10;

(b) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations, e.g., when determined by the method described in Example 10;

(c) the total number of viable cells in the combined first and third populations is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the resulting population, e.g., when determined by the method described in Example 10;

(d) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10; or

(e) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the second population, e.g., when determined by the method described in Example 10.

In some embodiments, in step (ii), the population of cells is contacted with a first viral vector at a first MOI and the population of cells is contacted with a second viral vector at a second MOI, such that the resulting population of cells comprises:

(a) the total number of viable cells in the combined second and third populations is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10;

(b) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations, e.g., when determined by the method described in Example 10;

(c) the total number of viable cells in the combined first and third populations is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the resulting population, e.g., when determined by the method described in Example 10;

(d) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10; or

(e) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the second population, e.g., when determined by the method described in Example 10.

In some embodiments, in step (ii), the population of cells is contacted with:

(a) a first viral vector at an MOI of from about 1 to about 10 (e.g., from about 2 to about 9, from about 3 to about 8, from about 4 to about 7, from about 5 to about 6, from about 1 to about 8, from about 1 to about 6, from about 1 to about 4, from about 8 to about 10, from about 6 to about 10, from about 4 to about 10, from about 1 to about 3, from about 2 to about 4, from about 3 to about 5, from about 4 to about 6, from about 5 to about 7, from about 6 to about 8, from about 7 to about 9, from about 8 to about 10, from about 2.5 to about 5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10);

(b) a second viral vector at an MOI of from about 0.1 to about 5 (e.g., from about 0.2 to about 4, from about 0.3 to about 3, from about 0.4 to about 2, from about 0.5 to about 1, from about 0.6 to about 0.9, from about 0.7 to about 0.8, from about 0.1 to about 4, from about 0.1 to about 3, from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 4 to about 5, from about 3 to about 5, from about 2 to about 5, from about 1 to about 5, from about 0.5 to about 5, from about 0.2 to about 5, from about 0.1 to about 0.5, from about 0.2 to about 1, from about 0.5 up to about 2, from about 1 to about 3, from about 2 to about 4, from about 3 to about 5, from about 0.5 to about 1, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5);

(c) a first viral vector at an MOI that is at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the MOI at which the population of cells is contacted with a second viral vector, or at least about 1-fold (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100-fold, such as about 2 to about 50-fold, about 3 to 20-fold, about 5 to about 15-fold, or about 8 to about 10-fold) greater therefrom; and/or

(d) a second viral vector at an MOI that is no more than 1/X, where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100, of the MOI at which the cell population is contacted with the first viral vector.

In some embodiments, the population of cells is contacted with the first viral vector at an MOI of about 2.5 to about 5. In some embodiments, the population of cells is contacted with the second viral vector at an MOI of about 0.5 to about 1.0. In some embodiments, the MOI for the first viral vector is about 8 to about 10 times higher than the MOI at which the population of cells is contacted with the second viral vector. In some embodiments, the MOI for the second viral vector is no more than 1/X, where X is 6, 8, 10, or 12, of the MOI at which the population of cells is contacted with the first viral vector.

In some embodiments, in step (ii), the population of cells is contacted with:

(a) a first viral vector at an MOI of from about 4 to about 5 (e.g., about 4.75); and/or

(b) a second viral vector at an MOI of from about 0.2 to about 1 (e.g., about 0.5).

In some embodiments, in step (ii), the population of cells comprises from about 1×10 ⁸ to about 5×10 ⁹ (e.g., from about 2×10 ⁸ to about 2×10 ⁹ or from about 4×10 ⁸ to about 1×10 ⁹ ) total viable cells. In some embodiments, the cells are suspended in culture at a concentration of from about 1×10 ⁶ to about 1×10 ⁷ (e.g., from about 2×10 ⁶ to about 5×10 ⁶ or from about 3×10 ⁶ to about 4×10 ⁶ ) viable cells/mL.

In some embodiments, an agent that stimulates a CD3/TCR complex is an agent that stimulates CD3. In some embodiments, an agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, an agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, an agent that stimulates a CD3/TCR complex is selected from an antibody (e.g., a single domain antibody (e.g., a single heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule is selected from an antibody (e.g., a single domain antibody (e.g., a single heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a CD3/TCR complex does not comprise a bead. In some embodiments, the agent that stimulates a costimulatory molecule does not comprise a bead. In some embodiments, the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody. In some embodiments, the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates the costimulatory molecule comprises an antibody to CD28 covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates the CD3/TCR complex and the agent that stimulates the costimulatory molecule comprise TransAct™ for T cells.

In some embodiments, the agent that stimulates the CD3/TCR complex does not contain a hydrogel. In some embodiments, the agent that stimulates the costimulatory molecule does not contain a hydrogel. In some embodiments, the agent that stimulates the CD3/TCR complex does not contain an alginate. In some embodiments, the agent that stimulates the costimulatory molecule does not contain an alginate.

In some embodiments, the agent that stimulates the CD3/TCR complex comprises a hydrogel. In some embodiments, the agent that stimulates the costimulatory molecule comprises a hydrogel. In some embodiments, the agent that stimulates the CD3/TCR complex comprises an alginate. In some embodiments, the agent that stimulates the costimulatory molecule comprises an alginate. In some embodiments, the agent that stimulates the CD3/TCR complex or the agent that stimulates the costimulatory molecule comprises MagCloudz™ from Quad Technologies.

In some embodiments, step (i) provides an increase in the percentage of cells expressing the CAR in the cell population of step (iii), such that the cell population of step (iii) exhibits a higher percentage of cells expressing the CAR (e.g., at least 10, 20, 30, 40, 50, or 60% higher) compared to cells obtained by an otherwise similar method without step (i).

In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (iii) is the same as the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (iii) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (iii) is no more than 5% or 10% different from the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (i).

In some embodiments, the population of cells from step (iii) exhibits a higher percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher), compared to cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the population of cells from step (iii) exhibits a higher percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher), compared to cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g. T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (iii) is the same as the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (iii) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (iii) differs by no more than 5% or 10% from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (i).

In some embodiments, the population of cells from step (iii) exhibits a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% lower), compared to cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the population of cells from step (iii) exhibits a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% lower), compared to cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the cell population of step (iii) is increased compared to the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of CAR-expressing memory T stem cells, such as CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the cell population of step (iii) is increased compared to the percentage of CAR-expressing memory T stem cells, such as CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, among cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the percentage of CAR-expressing memory T stem cells, such as CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing memory T stem cells, such as CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, among cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, among cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g. T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8 or 9 days. In some embodiments, the percentage of CAR-expressing memory T stem cells, such as CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the cell population of step (iii) is higher than the percentage of CAR-expressing memory T stem cells, such as CAR-expressing CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the cell population (e.g. T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8 or 9 days.

In some embodiments, the median gene set score (upregulated in TEM relative to downregulated in TSCM) for the cell population from step (iii) is approximately the same as, or differs from, the median gene set score (upregulated in TEM relative to downregulated in TSCM) for the cell population at the beginning of step (i) by no more than (e.g., increased by no more than) about 25, 50, 75, 100, or 125%. In some embodiments, the median gene set score (upregulated in TEM relative to downregulated in TSCM) for the population of cells from step (iii) is lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median gene set score (upregulated in TEM relative to downregulated in TSCM) for cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median gene set score (upregulated in TEMs relative to downregulated in TSCM) for the cell population of step (iii) is lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median gene set score (upregulated in TEMs relative to downregulated in TSCM) for cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the cell population (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days. In some embodiments, the median gene set score (upregulated in Tregs relative to downregulated in Teff) for the cell population from step (iii) is approximately the same as, or differs from, by no more than (e.g., increased by no more than) about 25, 50, 100, 150, or 200%. In some embodiments, the median gene set score (upregulated in Tregs relative to downregulated in Teff) for the population of cells from step (iii) is lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median gene set score (upregulated in Tregs relative to downregulated in Teff) for cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median gene set score (upregulated in Tregs relative to downregulated in Teff) for the cell population of step (iii) is lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median gene set score (upregulated in Tregs relative to downregulated in Teff) for cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the cell population (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days. In some embodiments, the median gene set score (repressed in stemness) for the population of cells from step (iii) is approximately the same as, or differs from, the median gene set score (repressed in stemness) for the population of cells at the beginning of step (i) by no more than (e.g., increased by no more than) about 25, 50, 100, 150, 200, or 250%. In some embodiments, the median gene set score (repressed in stemness) for the population of cells from step (iii) is lower (e.g., at least about 50, 100, or 125% lower) than the median gene set score (repressed in stemness) for cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median gene set score (repressed in stemness) for the population of cells from step (iii) is lower (such as at least about 50, 100, or 125% lower) than the median gene set score (repressed in stemness) for cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days. In some embodiments, the median gene set score (hypoxia-activated) for the population of cells from step (iii) is about the same as, or different from, (such as increased by, about 125, 150, 175, or 200%) the median gene set score (hypoxia-activated) for the population of cells at the beginning of step (i). In some embodiments, the median gene set score (hypoxia upregulated) for the population of cells from step (iii) is lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median gene set score (hypoxia upregulated) for cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median gene set score (activated by hypoxia) for the population of cells from step (iii) is lower (such as at least about 40, 50, 60, 70, or 80% lower) than the median gene set score (activated by hypoxia) for cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days. In some embodiments, the median gene set score (activated by autophagy) for the population of cells from step (iii) is about the same as, or differs from, by no more than (such as increased by no more than) about 180, 190, 200, or 210%. In some embodiments, the median gene set score (activated by autophagy) for the population of cells from step (iii) is lower (e.g., at least about 20, 30, or 40% lower) than the median gene set score (activated by autophagy) for cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median gene set score (activated by autophagy) for the population of cells from step (iii) is lower (e.g., at least 20, 30, or 40% lower) than the median gene set score (activated by autophagy) for cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the population of cells from step (iii), after incubation with a cell expressing an antigen recognized by the CAR, secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) than cells obtained by an otherwise similar process wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or cells obtained by an otherwise similar process that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the population of cells from step (iii) after administration in vivo persists longer or expands at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells obtained by an otherwise similar method in which step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the population of cells from step (iii) after administration in vivo persists longer or expands at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the population of cells from step (iii), upon administration in vivo, exhibits more potent anti-tumor activity (e.g., more potent anti-tumor activity at a low dose, e.g., a dose of no more than 0.15× ¹⁰⁶ , 0.2× ¹⁰⁶ , 0.25× ¹⁰⁶ , or 0.3× ¹⁰⁶ viable CAR-expressing cells) than cells obtained by an otherwise similar method wherein step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or cells obtained by an otherwise similar method that further comprises, after step (ii) and before step (iii), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8 or 9 days.

In some embodiments, the cell population of step (iii) is not expanded, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (i). In some embodiments, the cell population of step (iii) is reduced compared to the number of living cells in the cell population at the beginning of step (i), such as as assessed by the number of living cells. In some embodiments, the cell population of step (iii) is expanded with an increase in number by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, such as no more than 10%, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (i). In some embodiments, the population of cells from step (iii) is not expanded or is expanded in less than 0.5, 1, 1.5, or 2 hours, such as less than 1 or 1.5 hours, compared to the population of cells at the start of step (i).

In some embodiments, steps (i) and (ii) are performed in cell media (e.g., serum-free media) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-6 (e.g., IL-6/sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, steps (i) and (ii) are performed in cell media (e.g., serum-free media) comprising IL-7, IL-21, or a combination thereof. In some embodiments, steps (i) and (ii) are performed in cell media (e.g., serum-free media) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6/sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, step (i) is performed in cell media (e.g., serum-free media) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-6 (e.g., IL-6/sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, step (ii) is performed in cell media (e.g., serum-free media) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-6 (e.g., IL-6/sIL-6Ra), an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, step (i) is performed in cell media (e.g., serum-free media) comprising IL-7, IL-21, or a combination thereof. In some embodiments, step (ii) is performed in cell media (e.g., serum-free media) comprising IL-7, IL-21, or a combination thereof. In some embodiments, step (i) is performed in cell media (e.g., serum-free media) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6/sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, step (ii) is performed in cell media (e.g., serum-free media) comprising IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6/sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, the cell media is a serum-free medium comprising a serum substitute. In some embodiments, the serum substitute is CTS™ Immune Cell Serum Replacement (ICSR).

In some embodiments, the above methods further comprise, prior to step (i) (iv), obtaining fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or removal product (e.g., fresh thymectomy product)), from an organization, such as a laboratory, hospital, or healthcare facility.

In some embodiments, the above methods further comprise, prior to step (i) (v), isolating the population of cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells) contacted in step (i) from a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or removal product (e.g., a fresh thymectomy product)). In some embodiments, step (iii) is performed no later than 35 hours after the start of step (v), such as no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), such as no later than 30 hours after the start of step (v). In some embodiments, the cell population of step (iii) is not expanded or is expanded by an increase in number of no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of living cells, compared to the cell population at the end of step (v).

In some embodiments, the above methods further comprise, prior to step (i), obtaining cryopreserved T cells isolated from the leukapheresis product (or an alternative source of hematopoietic tissue, such as cryopreserved T cells isolated from whole blood, bone marrow, or a product obtained from a biopsy or removal of a tumor or organ (e.g., thymectomy)), from an organization, such as a laboratory, hospital, or healthcare facility.

In some embodiments, the above methods further comprise, prior to step (i) (iv), obtaining a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved product of whole blood, a cryopreserved product of bone marrow, or a cryopreserved product obtained from a biopsy or removal of a tumor or organ (e.g., a cryopreserved product obtained from a thymectomy)), from an organization, such as a laboratory, hospital, or healthcare facility.

In some embodiments, the above methods further comprise, prior to step (i) (v), isolating the population of cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells) contacted in step (i) from the cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved product comprising whole blood, a cryopreserved product comprising bone marrow, or a cryopreserved product obtained from a biopsy or removal of a tumor or organ (e.g., a cryopreserved product obtained from a thymectomy)). In some embodiments, step (iii) is performed no later than 35 hours after the start of step (v), such as no later than 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), such as no later than 30 hours after the start of step (v). In some embodiments, the cell population of step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of live cells, compared to the cell population at the end of step (v).

In some embodiments, the cells of step (iii) are cultured for about two to about four days, such as about three days (e.g., about 72 hours after harvest), before measuring the level of CAR expression in a portion thereof (e.g., before measuring the percentage of viable cells expressing the CAR in a portion thereof, such as before measuring the percentage of viable cells expressing the CAR for BCMA in a portion thereof). In some embodiments, measuring CAR expression is performed about 4 days (e.g., 96 hours) after step (ii). In some embodiments, the level of CAR expression is measured by flow cytometry.

In some embodiments, the invention features a method of producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising (1) contacting the population of cells (e.g., T cells, e.g., T cells isolated from a frozen leukapheresis product) with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6, or a combination thereof, (2) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding the CAR, thereby producing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (3) collecting the population of cells (e.g., T cells) for storage (e.g., reconstituting the population of cells in cryopreservation medium) or administration, wherein (a) step (2) is performed together with step (1) or no later than 5 hours after the start of step (1), such as no later than 1, 2, 3, 4 or 5 hours after the start of step (1), and step (3) is performed no later than 26 hours after the start of step (1), such as no later than 22, 23, 24 or 25 hours after the start of step (1), such as no later than 24 hours after the start of step (1), or (b) the cell population of step (3) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of live cells, compared to the cell population at the start of step (1). In some embodiments, the nucleic acid molecule of step (2) is a DNA molecule. In some embodiments, the nucleic acid molecule of step (2) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (2) is in a viral vector, such as a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (2) is in a non-viral vector. In some embodiments, the nucleic acid molecule of step (2) is in a plasmid. In some embodiments, the nucleic acid molecule of step (2) is not in any vector. In some embodiments, step (2) involves transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR. In some embodiments, the nucleic acid molecule encoding a CAR is a nucleic acid molecule as disclosed herein. In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR. In some embodiments, the first and second nucleic acid sequences are located on a single nucleic acid molecule, such as where the first nucleic acid sequence and the second nucleic acid sequence are separated by a third nucleic acid sequence encoding a self-cleavage site (e.g., a P2A site, a T2A site, an E2A site, or an F2A site). In some embodiments, the first and second nucleic acid sequences are located on separate nucleic acid molecules. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a CAR, wherein the CAR comprises a first VH (VH1), a first VL (VL1), a second VH (VH2), a second VL (VL2), a transmembrane domain, and an intracellular signaling domain, wherein VH1 and VL1 bind a first antigen and VH2 and VL2 bind a second antigen, wherein VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optional Linker 1 ("L1") - VH2 - optional Linker 2 ("L2") - VL2 - optional Linker 3 ("L3") - VL1, VH1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VL1, VL1 - optional L1 - VH2 - optional L2 - VL2 - optional L3 - VH1, VL1 - optional L1 - VL2 - optional L2 - VH2 - optional L3 - VH1, VH2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VL2, VH2 - optional L1 - VL1 - optional L2 - VH1 - optional L3 - VL2, VL2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VH2 or VL2 - optional L1 - VL1 - optional L2 - VH1 - optional L3 - VH2.

In some embodiments, step (2) is performed together with step (1). In some embodiments, step (2) is performed no later than 5 hours after the start of step (1). In some embodiments, step (2) is performed no later than 1, 2, 3, 4, or 5 hours after the start of step (1). In some embodiments, step (3) is performed no later than 26 hours after the start of step (1). In some embodiments, step (3) is performed no later than 22, 23, 24, or 25 hours after the start of step (1). In some embodiments, step (3) is performed no later than 24 hours after the start of step (1).

In some embodiments, the cell population of step (3) is not expanded, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (1). In some embodiments, the cell population of step (3) is expanded by an increase in abundance of no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (1). In some embodiments, the cell population of step (3) is expanded by an increase in abundance of no more than 10%, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (1).

In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-7. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) and IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-21 and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) and IL-21.

In some embodiments, the population of cells from step (3) exhibits a higher percentage of naive cells among the cells expressing the CAR (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) compared to cells obtained by an otherwise similar method that further comprises contacting the population of cells with, e.g., an anti-CD3 antibody.

In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population from step (3) is the same as the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (3) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (3) is no more than 5% or 10% different from the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (3) is increased compared to the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (3) is increased by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population of step (3) is increased by at least 10% or 20% compared to the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in the cell population at the beginning of step (1).

In some embodiments, the population of cells from step (3) exhibits a higher percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher), compared to cells obtained by an otherwise similar method wherein step (3) is performed more than 26 hours after the start of step (1), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the population of cells from step (3) exhibits a higher percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher), compared to cells obtained by an otherwise similar method that further comprises, after step (2) and before step (3), expanding the population of cells (e.g. T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (3) is the same as the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (3) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (3) differs by no more than 5% or 10% from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (3) is reduced compared to the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (3) is reduced by at least 10% or 20% compared to the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (1). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population of step (3) is reduced by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% compared to the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the beginning of step (1).

In some embodiments, the population of cells from step (3) exhibits a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% lower), compared to cells obtained by an otherwise similar method wherein step (3) is performed more than 26 hours after the start of step (1), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the population of cells from step (3) exhibits a lower percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% lower), compared to cells obtained by an otherwise similar method that further comprises, after step (2) and before step (3), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the population of cells from step (3) after administration in vivo persists longer or expands at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells obtained by an otherwise similar method in which step (3) is performed more than 26 hours after the start of step (1), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the population of cells from step (3) after administration in vivo persists longer or expands at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells obtained by an otherwise similar method that further comprises, after step (2) and before step (3), expanding the population of cells (e.g., T cells) in vitro for more than 3 days, such as for 5, 6, 7, 8, or 9 days.

In some embodiments, the cell population of step (3) is not expanded, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (1). In some embodiments, the cell population of step (3) is expanded by an increase in abundance of no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (1). In some embodiments, the cell population of step (3) is expanded by an increase in abundance of no more than 10%, such as as assessed by the number of living cells, compared to the cell population at the beginning of step (1). In some embodiments, the number of living cells in the cell population from step (3) is reduced compared to the number of living cells in the cell population at the beginning of step (1), such as as assessed by the number of living cells.

In some embodiments, the cell population of step (3) is not expanded compared to the cell population at the beginning of step (1), for example, as assessed by the number of living cells. In some embodiments, the cell population of step (3) is expanded in less than 0.5, 1, 1.5, or 2 hours, for example, less than 1 or 1.5 hours, compared to the cell population at the beginning of step (1).

In some embodiments, the cell population is not contacted in vitro with an agent that stimulates the CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the cell surface, or if contacted, the duration of the contacting step is less than 2 hours, such as no more than 1 or 1.5 hours. In some embodiments, the agent that stimulates the CD3/TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody). In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3/TCR complex or the agent that stimulates the costimulatory molecule is selected from an antibody (e.g., a single domain antibody (e.g., a single heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand).

In some embodiments, steps (1) and/or (2) are performed in cell media containing no more than 5, 4, 3, 2, 1, or 0% serum. In some embodiments, steps (1) and/or (2) are performed in cell media containing no more than 2% serum. In some embodiments, steps (1) and/or (2) are performed in cell media containing about 2% serum. In some embodiments, steps (1) and/or (2) are performed in cell media containing an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (1) is performed in cell media containing no more than 5, 4, 3, 2, 1, or 0% serum. In some embodiments, step (1) is performed in cell media containing no more than 2% serum. In some embodiments, step (1) is performed in cell media containing about 2% serum. In some embodiments, step (2) is performed in cell media containing no more than 5, 4, 3, 2, 1, or 0% serum. In some embodiments, step (2) is performed in cell media containing no more than 2% serum. In some embodiments, step (2) is performed in cell media containing about 2% serum. In some embodiments, step (1) is performed in cell media containing an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (2) is performed in cell media containing an LSD1 inhibitor or a MALT1 inhibitor.

In some embodiments, the above methods further comprise, prior to step (i) (iv), obtaining fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or removal product (e.g., fresh thymectomy product)), from an organization, such as a laboratory, hospital, or healthcare facility.

In some embodiments, the above methods further comprise, prior to step (i) (v), isolating the population of cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells) contacted in step (i) from a fresh leukapheresis product (or an alternative source of hematopoietic tissue, such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or removal product (e.g., a fresh thymectomy product)). In some embodiments, step (iii) is performed no later than 35 hours after the start of step (v), such as no later than 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), such as no later than 30 hours after the start of step (v). In some embodiments, the cell population of step (iii) is not expanded or is expanded by an increase in number of no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of living cells, compared to the cell population at the end of step (v).

In some embodiments, the above methods further comprise, prior to step (i), obtaining cryopreserved T cells isolated from the leukapheresis product (or an alternative source of hematopoietic tissue, such as cryopreserved T cells isolated from whole blood, bone marrow, or a product obtained from a biopsy or removal of a tumor or organ (e.g., thymectomy)), from an organization, such as a laboratory, hospital, or healthcare facility.

In some embodiments, the above methods further comprise, prior to step (i) (iv), obtaining a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved product of whole blood, a cryopreserved product of bone marrow, or a cryopreserved product obtained from a biopsy or removal of a tumor or organ (e.g., a cryopreserved product obtained from a thymectomy)), from an organization, such as a laboratory, hospital, or healthcare facility.

In some embodiments, the above methods further comprise, prior to step (i) (v), isolating the population of cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells) contacted in step (i) from the cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved product comprising whole blood, a cryopreserved product comprising bone marrow, or a cryopreserved product obtained from a biopsy or removal of a tumor or organ (e.g., a cryopreserved product obtained from a thymectomy)). In some embodiments, step (iii) is performed no later than 35 hours after the start of step (v), such as no later than 27, 28, 29, 30, 31, 32, 33, 34 or 35 hours after the start of step (v), such as no later than 30 hours after the start of step (v). In some embodiments, the cell population of step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of live cells, compared to the cell population at the end of step (v).

In some embodiments, the cell population at the beginning of step (i) or step (1) is enriched in IL6R expressing cells (e.g., cells that are IL6Rα and/or IL6Rβ positive). In some embodiments, the cell population at the beginning of step (i) or step (1) comprises at least 40%, 45%, 50%, 55%, 60%, 65%, or 70% IL6R expressing cells (e.g., cells that are IL6Rα and/or IL6Rβ positive).

In some embodiments, steps (i) and (ii) or steps (1) and (2) are performed in cell media containing IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, IL-15 increases the ability of a cell population to proliferate, such as after 10, 15, 20, or 25 days. In some embodiments, IL-15 increases the percentage of IL6Rβ-expressing cells in a cell population.

In some embodiments of the above methods, the methods are performed in a closed system. In some embodiments, all of the separation, activation, transduction, incubation, and washing of the T cells are performed in a closed system. In some embodiments of the above methods, the methods are performed in separate devices. In some embodiments, the separation, activation, transduction, incubation, and washing of the T cells are performed in separate devices.

In some embodiments of the above methods, the methods further comprise adding an adjuvant or transduction enhancing reagent to the cell culture medium to enhance transduction efficiency. In some embodiments, the adjuvant or transduction enhancing reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction enhancing reagent is selected from LentiBOOST™ (Sirion Biotech), Vectofusin-1, F108, Hexadimethrine Bromide (Polybrene), PEA, Pluronic F68, Pluronic F127, Synperonic, or LentiTrans™. In some embodiments, the adjuvant is LentiBOOST™ (Sirion Biotech).

In some embodiments of the above methods, transducing a population of cells (e.g., T cells) with a viral vector comprises exposing the population of cells and the viral vector to centrifugal force under conditions that enhance transduction efficiency. In one embodiment, the cells are transduced by spinoculation.

In some embodiments of the above methods, cells (e.g., T cells) are activated and transduced in a cell culture flask containing a gas-permeable membrane at the base that allows for handling large volumes of media without significantly disrupting gas exchange. In some embodiments, cell growth is achieved by providing access, e.g., substantially continuous access, to nutrients via convection.

In some embodiments of the above methods, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

In some embodiments, the antigen binding domain binds to an antigen selected from CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (e.g., ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, hsp 70-2 mutant, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC. In some embodiments, the antigen-binding domain comprises a CDR, VH, VL, scFv, or CAR sequence disclosed herein. In some embodiments, the antigen-binding domain comprises a VH and VL, wherein the VH and VL are connected by a linker, wherein the linker optionally comprises the amino acid sequence of SEQ ID NO: 63 or 104.

In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from a T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the intracellular signaling domain comprises a primary signaling domain. In some embodiments, the primary signaling domain provides a functional signaling domain derived from CD3zeta, TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d. In some embodiments, the primary signaling domain provides a functional signaling domain derived from CD3zeta. In some embodiments, the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain provides a functional signaling domain derived from a class I MHC molecule, a TNF protein receptor, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an NK cell activating receptor, BTLA, a Toll-like receptor ligand, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD28-OX40, CD28-4-1BB, or a ligand that specifically binds to CD83. In some embodiments, the costimulatory signaling domain provides a functional signaling domain derived from 4-1BB. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory signaling domain, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3zeta. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the present invention describes a population of CAR-expressing cells (e.g., autologous or allogeneic T cells or NK cells expressing a CAR) obtained by any of the above methods or any other method disclosed herein. In some embodiments, disclosed herein is a pharmaceutical composition comprising a population of CAR-expressing cells disclosed herein and a pharmaceutically acceptable carrier.

In some embodiments, the population comprises:

(a) the first population of cells containing CAR for BCMA but not CAR for CD19;

(b) a second population of cells containing a CAR for CD19 but not a CAR for BCMA; and

(c) a third population of cells containing both CAR for BCMA and CAR for CD19.

In some embodiments:

(i) the total number of viable cells in the combined second and third populations is less than or equal to approximately 110% (e.g., less than or equal to approximately 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations;

(ii) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations; and/or

(iii) the total number of viable cells in the combined first and third populations is greater than or equal to approximately 5% (e.g., greater than or equal to approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the population.

In some embodiments, the population further comprises a fourth population of cells that do not contain CAR.

In some embodiments:

(i) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations;

(ii) the total number of viable cells in the second population is less than or equal to from about 45% to about 50% (e.g., about 47%); from about 50% to about 55% (e.g., about 53%); from about 60% to about 65% (e.g., about 63%); or from about 80% to about 85% (e.g., about 82%) of the total number of viable cells in the combined first and third populations.

In some embodiments, in the final CAR cell product manufactured using the methods described herein, the total amount of beads (e.g., anti-CD4 beads, anti-CD8 beads, and/or TransAct beads) is no more than 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5% of the total amount of beads added during the manufacturing method.

In some embodiments, the present invention features a population of cells expressing a CAR (e.g., autologous or allogeneic T cells or NK cells expressing a CAR) comprising one or more of the following characteristics: (a) about the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO- CCR7+ T cells, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO- CCR7+ cells, in the same population of cells before they were engineered to express a CAR; (b) a change in the number of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, of from about 5% to about 10%, such as compared to the percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, in the same population of cells before they were engineered to express the CAR; (c) an increased percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, such as increased by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold, compared to the percentage of naive cells, such as naive T cells, such as CD45RO- CCR7+ T cells, in the same population of cells prior to engineering them to express the CAR; (d) approximately the same percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (e) a change in the number of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, of from about 5% to about 10% compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (f) a reduced percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, such as reduced by at least 20, 25, 30, 35, 40, 45 or 50%, compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+ CD45RO+ T cells, in the same population of cells before they were engineered to express the CAR; (g) approximately the same percentage of memory T stem cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of memory T stem cells, e.g., CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same cell population before they were engineered to express the CAR; (h) a change in the number of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, of between about 5% and about 10%, compared to the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells prior to their engineering to express the CAR; or (i) an increased percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, compared to the percentage of memory T stem cells, such as CD45RA+ CD95+ IL-2 receptor β+ CCR7+ CD62L+ T cells, in the same population of cells before they were engineered to express the CAR.

In some embodiments, the present invention features a population of cells expressing a CAR (e.g., autologous or allogeneic T cells or NK cells expressing a CAR), wherein (a) the median gene set score (upregulated in TEMs relative to downregulated in TSCM) for the population of cells is approximately the same as, or different from (e.g., increased by) about 25, 50, 75, 100, or 125%; (b) the median gene set score (upregulated in Tregs relative to downregulated in Teff) for the population of cells is approximately the same as, or differs from, the median gene set score (upregulated in Tregs relative to downregulated in Teff) for the population of cells prior to being engineered to express the CAR by no more than about 25, 50, 100, 150, or 200%; (c) the median gene set score (downregulated in stemness) for the population of cells is approximately the same as, or differs from, the median gene set score (downregulated in stemness) for the population of cells prior to being engineered to express the CAR by no more than about 25, 50, 100, 150, 200, or 250%; (d) the median gene set score (activated by hypoxia) for the population of cells is approximately the same as, or differs from, the median gene set score (activated by hypoxia) for the population of cells prior to being engineered to express the CAR by at most (e.g., increased by at most) about 125, 150, 175, or 200%; or (e) the median gene set score (activated by autophagy) for the population of cells is approximately the same as, or differs from, the median gene set score (activated by autophagy) for the population of cells prior to being engineered to express the CAR by at most (e.g., increased by at most) about 180, 190, 200, or 210%.

In some embodiments, the present invention describes a method for enhancing an immune response in a subject, comprising administering to the subject a population of cells expressing a CAR disclosed herein or a pharmaceutical composition disclosed herein, thereby enhancing the immune response in the subject.

In some embodiments, disclosed herein is a method of treating cancer in a subject comprising administering to the subject a population of cells expressing a CAR disclosed herein or a pharmaceutical composition disclosed herein, thereby treating the cancer in the subject. In some embodiments, the cancer is a solid cancer, such as one or more of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer or metastases thereof. In some embodiments, the cancer is a hematological malignancy, such as one selected from chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal mucosal lymphoid tissue marginal zone lymphoma), marginal zone cell lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone cell lymphoma, splenic lymphoma/leukemia, diffuse small B-cell lymphoma of the splenic red pulp, variant hairy cell leukemia, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, nodular marginal zone cell lymphoma, pediatric nodular marginal zone cell lymphoma, primary cutaneous follicular center cell lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma.

In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is an anti-cancer therapeutic agent, such as a chemotherapy, radiation therapy, or immunoregulatory therapy. In some embodiments, the second therapeutic agent is IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)).

In some embodiments, provided herein is an isolated cell or population of cells obtained by a method described herein, comprising one or more cells comprising:

(a) a first nucleic acid molecule encoding a first CAR that comprises a BCMA binding domain, a first transmembrane domain and a first intracellular signalling domain, wherein the BCMA binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC) and a heavy chain complementarity determining region 3 (CDR3 HC), and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC) and a light chain complementarity determining region 3 (CDR3 LC), wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LCs contain amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96 and 97, respectively; and

(b) a second nucleic acid molecule encoding a second CAR that comprises a CD19 binding domain, a second transmembrane domain and a second intracellular signaling domain, wherein the CD19 binding domain comprises a VH comprising CDR1 HC, CDR2 HC and CDR3 HC, and a VL comprising CDR1 LC, CDR2 LC and CDR3 LC, wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 295, 304 and 297-300, respectively.

In some embodiments, provided herein is an isolated cell comprising:

(a) a first nucleic acid molecule encoding a first CAR that comprises a BCMA binding domain, a first transmembrane domain and a first intracellular signalling domain, wherein the BCMA binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC) and a heavy chain complementarity determining region 3 (CDR3 HC), and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC) and a light chain complementarity determining region 3 (CDR3 LC), wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LCs contain amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96 and 97, respectively; and

(b) a second nucleic acid molecule encoding a second CAR that comprises a CD19 binding domain, a second transmembrane domain and a second intracellular signaling domain, wherein the CD19 binding domain comprises a VH comprising CDR1 HC, CDR2 HC and CDR3 HC, and a VL comprising CDR1 LC, CDR2 LC and CDR3 LC, wherein CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 295, 304 and 297-300, respectively.

In some embodiments, the VH and VL of the BCMA binding domain comprise the amino acid sequences of SEQ ID NOs: 93 and 102, respectively. In some embodiments, the VH and VL of the CD19 binding domain comprise the amino acid sequences of SEQ ID NOs: 250 and 251, respectively. In some embodiments, the VH and VL of the BCMA binding domain comprise the amino acid sequences of SEQ ID NOs: 93 and 102, respectively, and the VH and VL of the CD19 binding domain comprise the amino acid sequences of SEQ ID NOs: 250 and 251, respectively. In some embodiments, the BCMA binding domain comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the BCMA binding domain comprises the amino acid sequence of SEQ ID NO: 105 and the CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the second CAR comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 107; and the second CAR comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 259, 258, or 416. In some embodiments, the second CAR is encoded by the nucleic acid sequence of SEQ ID NO: 417, 355, 356, or 354. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 259, 258, or 416, and the second CAR is encoded by the nucleic acid sequence of SEQ ID NO: 417, 355, 356, or 354.

In some embodiments, provided herein is a pharmaceutical composition comprising a cell or population of cells described herein.

In some embodiments, provided herein is a method of providing anti-tumor immunity in a subject or treating a subject having a disease associated with BCMA expression, comprising administering to the subject an effective amount of a cell or population of cells or a pharmaceutical composition described herein.

In some embodiments, the disease associated with BCMA expression is a hematological cancer or a solid cancer, such as a hematological cancer or a solid cancer described herein.

In some embodiments, the disease is selected from acute leukemia, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorders (e.g., asymptomatic myeloma ("smoldering" multiple myeloma or smoldering myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloidosis in the form of light chain amyloidosis or POEMS syndrome (also known as POEMS syndrome) Crowe-Fukasa, Takatsuki disease and PEP syndrome)) or their combinations.

In some embodiments, the disease is multiple myeloma.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other literature references (e.g., sequence identification numbers in databases) cited herein are incorporated by reference in their entireties. For example, all sequences from GenBank, Unigene, and Entrez cited herein, such as in any table herein, are incorporated by reference. Where multiple sequence accession numbers are referenced for a single gene or protein, all sequence variants are covered.

In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. Headings, subheadings or numbered or lettered elements, such as (a) (b) (i), etc., are provided solely for ease of reading. The use of headings or numbered or lettered elements in this document does not require that the steps or elements be in alphabetical order or that the steps or elements are necessarily separated from each other. Other features, objects and advantages of the present invention will be apparent from the description and drawings, as well as from the claims.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1A-1H . Jurkat NFAT-luciferase reporter cell assay (JNL) with an automated system was used to test the function of CAR for BCMA. CAR clones were evaluated in the JNL reporter cell assay for antigen-dependent activity. JNL cells containing the indicated CAR clones or untransduced JNL cells (UTD) were co-cultured with medium alone (Fig. 1G and 1H) or with target cell lines (KMS11 as a BCMA-positive cell line (Fig. 1A and 1C) and NALM6 as a BCMA-negative cell line (Fig. 1E and 1F)) at different ratios, and luciferase activity was measured as luminescence intensity. Clones were considered active if the luminescence intensity was 2-fold higher than that of UTD cells in the presence of antigen-expressing cells. The luminescence reading is a direct measure of CAR stimulation. Fig. 1B and 1D are graphs showing the expression level of BCMA CAR on JNL cells as determined by flow cytometry using recombinant human (r)BCMA_Fc-AF647. The 1x or 2x platform indicated 40,000 H293 cells or 80,000 H293 cells seeded for virus production.

Fig. 2 . Expression level of CAR for BCMA on primary human T cells. Cells were stained with human rBCMA_Fc-AF647 reagent and analyzed by flow cytometry. The percentage of CAR+ cells and MFI are plotted for day 5 and day 9 of cell culture. The data are summarized in Table 27, which includes the viral titer achieved for the respective CARs.

Fig. 3A-3C. The ability of T cells expressing the indicated CARs to mediate cell lysis and cytokine production was assessed against the firefly luciferase-expressing KMS11 cell line (KMS11-luc). Fig. 3A. CAR T cells were co-cultured with KMS11-luc target cells at the indicated E:T ratios. % cell killing was determined by the difference in luciferase signal between target cells without effector T cells (control) and with effector T cells (experiment), expressed as a percentage of control. UTD represents untransduced T cells. Fig. 3B. Background killing was observed for the BCMA-negative NALM6 cell line. Fig. 3C. IFNγ levels were measured by MSD in supernatants collected after 24 h from these co-culture systems at an E:T ratio of 2.5. All data are expressed as mean +/- standard deviation.

Fig. 4A-4C. CAR expression in T cells transduced at an MOI of 5 (virus titer was determined by the first CAR expressed in SupT1 cells). Fig. 4A is a table summarizing % CAR19, % CAR for BCMA, % double positive, % CAR19 only, and % CAR for BCMA only for the different constructs. Fig. 4B is a series of flow cytometry plots showing cell staining for BCMA CAR surface expression (x-axis) and CD19 CAR surface expression (y-axis). Fig. 4C is a pair of bar graphs showing MFI for BCMA CAR (upper panel) and MFI for CD19 CAR (lower panel).

Fig. 5A-5C: In vitro killing assay using CAR-T cells at day 8. Fig. 5A-5C are a series of graphs showing the % killing of BCMA-positive KMS11 cells, CD19-positive Nalm6 cells, or BCMA/CD19-negative cells, respectively, at the indicated E:T ratios.

Fig. 6A-6D: In vitro cytokine production assay using CAR-T cells at day 8. Fig. 6A-6D are a series of bar graphs showing IFN-gamma production by CAR-T cells when co-cultured with BCMA-positive KMS11 cells or CD19-positive Nalm6 cells.

Fig. 7A-7C. Individual CAR expression in cells made using the ARM method. Fig. 7A-7B are histograms showing the expression pattern of both the BCMA CAR and the CD19 CAR at 24 hours or 72 hours after transduction of primary human T cells made using the ARM method. The studies used an MOI of 1 based on the titer in SupT1 determined by the expression of the upstream CAR. In each of Fig. 7A and 7B, the left panel is a panel of histograms showing staining using rBCMA-Fc, and the right panel is a panel of histograms showing staining using an anti-idiotype antibody that binds to the CD19 CAR. Constructs #244 (“c244”) and #245 (“c245”) are a mono-CAR for CD19 and a mono-CAR for BCMA, respectively. Figure 7C is a panel of flow cytometry plots showing the expression pattern of the CAR for BCMA and CD19 72 hours after transduction of primary human T cells using an MOI of 1 based on the titer of the upstream CAR.

Fig. 8A-8C. In vivo antitumor activity of construct #236 ("c236") and construct #238 ("c238") using three mouse models: a KMS-11 (BCMA+ CD19-) disseminated multiple myeloma model expressing a luciferase reporter gene (KMS11-Luc) (Fig. 8A), a Nalm6-Luc (CD19+BCMA-) xenograft mouse model (Fig. 8B), and a mixed model of 95% KMS-luc cells and 5% NALM6-Luc cells (Fig. 8C). Tumor burden is expressed as whole body luminescence (photons/s) and is depicted as mean tumor burden+SEM. On day 7 or 8 post-tumor inoculation, mice were treated with c236 and c238 at the indicated doses of BCMA-CAR+ or CD19-CAR+ T cells (approximate numbers of viable CAR+ T cells) as shown in Table 30. Vehicle medium (PBS) and untransduced T cells (UTD) served as negative controls. The BCMA monoCAR PI61 and CD19 monoCAR CTL119 were also used as controls.

Fig. 9A-9C. Graft-versus-host disease-induced body weight loss. All mice were individually monitored for body weight loss, a detectable marker of X-GvHD, by measuring body weight over time. Body weight (BWT) was plotted as % change from baseline.

Fig. 10A-10C: In vivo expansion of peripheral blood CD3+ T cells was analyzed by flow cytometry for up to 4 weeks after infusion.

Fig. 11A-11C. Expansion of CAR+ T cellsin vivo(percentage of BCMA-CAR+) was analyzed by flow cytometry for up to 4 weeks after infusion.

Fig. 12A-12C. Expansion of CAR+ T cellsin vivo(CAR+ double counts) were analyzed by flow cytometry for up to 4 weeks post-infusion.

Fig. 13A-13C.Kinetic plasma IFNγ levelsin vivo. Plasma IFNγ levels in all three mouse models treated with c236 and c238, as well as mono-CAR controls at the corresponding CAR-T doses, were plotted. Mice were bled and plasma cytokine levels were measured by MSD assay.

Fig. 14A and 14B . In vivo potency and cell expansion of 236- and c238-derived cells in a xenograft mouse model of multiple myeloma. Fig. 14A . NSG mice were injected with the KMS11 multiple myeloma cell line that expressed a luciferase reporter gene. Tumor burden is expressed as whole-body luminescence (photons/s) and shown as mean tumor burden+SEM. On day 8 post-tumor inoculation, mice were treated with c236 and c238 at 9e4 twice BCMA/CD19-CAR+ T cells (approximate number of viable CAR+ T cells). Vehicle medium (PBS) and untransduced T cells (UTD) served as negative controls. Fig. 14B Peripheral blood CAR+ T cell expansion was analyzed by flow cytometry for up to 4 weeks post-infusion. Double expansion of BCMA/CD19-CAR+ T cells was observed in the Rx CAR-T c236 and c238 groups.

Fig. 15A and 15B . CAR expression in cells made using the ARM method. Flow cytometry plots showing CAR expression for BCMA and CD19 in double-positive cells 96 hours (Fig. 15A) and 7 days (Fig. 15B) after virus addition to human primary T cells made using the ARM method. Studies used an MOI of 2 based on the titer in SupT1 determined by CAR expression in double-positive cells (PI61 or R1G5 and CTL119 clones positive) detected with an anti-idiotype antibody binding to the CD19 CAR and recombinant BCMA_Fc (AF647) binding to PI61 or R1G5. Mono-BCMA-CAR-T PI61 and R1G5, as well as mono-CD19-CAR-T CTL119 served as controls.

Fig. 16 . CAR expression at day 7 in the TM method using an MOI of 5. Flow cytometry plots showing CAR expression for BCMA and CD19 in double-positive cells 7 days after virus addition to human primary T cells made using the TM method. Studies used an MOI of 5 based on the titer in SupT1 determined by CAR expression in double-positive cells (positive for PI61 or R1G5 and CTL119 clones) detected with an anti-idiotype antibody binding to the CAR for CD19 and recombinant BCMA_Fc (AF647) binding to PI61 or R1G5.

Fig. 17A and 17B . Specific killing of BCMA- or CD19-expressing tumor cells in vitro by T cells engineered with BCMA and CD19 CAR constructs in a diabody format. The ability of T cells expressing PI61/CTL119 clones to mediate cell lysis was assessed against the KMS11-Luc or NALM6-Luc target cell line. CAR T cells were co-cultured with BCMA+ KMS-11-luc target cells or BCMA− NALM6-Luc target cells at the indicated E:T ratios for 20 h, and % cell killing, defined as the difference in luciferase signal between target cells without effector T cells (control) and with effector T cells (experiment), expressed as a percentage of control, was measured as a surrogate for target cell lysis. UTD represents untransduced T cells. PI61 or CTL119 monovariants served as controls.

Fig. 18A and 18B . Cytokine production by T cells engineered with BCMA and CD19 CAR constructs in diabody format in response to BCMA- or CD19-expressing tumor cells. IFNγ (Fig. 18A) and IL-2 (Fig. 18B) levels were measured by MSD in co-culture supernatants from the 1.25:1 killing assay.

Fig. 19 . Percentages of double CAR-positive population, CAR-positive BCMA population, and CAR-positive CD19 population at day 4.

Fig. 20 . Flow cytometry graphs showing cell staining with rBCMA-Fc and an anti-idiotype antibody that binds to the CD19 CAR.

Fig. 21 . Percentages of total CAR-positive populations at day 4 and day 7 under the indicated conditions.

Fig. 22 . Cell counts (left panel) and percentage of viable cells (right panel) at days 0, 1, 3, and 7 under the indicated conditions.

Fig. 23A, 23B, and 23C. Single-cell RNA-seq data for parental cells (Fig. 23A), day 1 cells (Fig. 23B), and day 9 cells (Fig. 23C). "n Gene" plots show the number of expressed genes per cell. "n UMI" plots show the number of unique molecular identifiers (UMI) per cell.

Fig. 24A, 24B, 24C, and 24D. Nonlinear dimensionality reduction and multivariate visualization (TSNE) plots comparing baseline (Fig. 24A), day 1 (Fig. 24B), and day 9 (Fig. 24C) cells for a proliferation signature determined by the expression of CCNB1 , CCND1 , CCNE1 , PLK1 , and MKI67 genes. Each dot represents a cell in the sample. Cells shown in light gray do not express proliferative genes, whereas cells shaded in dark express one or more proliferative genes. Fig. 24D is a violin plot showing the distribution of gene set scores for the gene set consisting of genes that characterize the resting state of T cells relative to those that characterize the activated state of T cells for day 1 cells, day 9 cells, and naive cells. In Fig. 24D, a higher gene set score (activated in the resting state relative to repressed in the activated state) indicates an increase in the expression of the resting T cell phenotype, whereas a lower gene set score (activated in the resting state relative to repressed in the activated state) indicates an increase in the expression of the activated T cell phenotype. Naive cells were generally more resting compared to day 9 and day 1 cells. Day 1 cells exhibited the highest gene set score for activation.

Fig. 25A, 25B, 25C, 25D, and 25E. Gene set analysis for baseline, day 1, and day 9 cells. In Fig. 25A, a higher gene set score for the "upregulated in TEMs relative to downregulated in TSCMs" gene set indicates an increase in the effector memory T cell (TEM) phenotype in the sample, while a lower gene set score indicates an increase in the stem cell memory T cell (TSCM) phenotype. In Fig. 25B, a higher gene set score for the "upregulated in Tregs relative to downregulated in Teffs" gene set indicates an increase in the regulatory T cell (Treg) phenotype, while a lower gene set score indicates an increase in the effector T cell (Teff) phenotype. In Fig. 25C, a lower gene set score for the "downregulated in stemness" gene set indicates an increase in the stemness phenotype. In Fig. 25D, a higher gene set score for the "upregulated in hypoxia" gene set indicates an increase in the hypoxia-specific phenotype. In Fig. 25E, a higher gene set score for the "upregulated in autophagy" gene set indicates an increase in the autophagy-specific phenotype. Day 1 cells appeared similar to parental cells with respect to memory signature, stem cell-likeness, and differentiation. Day 9 cells, however, showed a higher degree of metabolic stress.

Figs. 26A, 26B, and 26C. Gene set analysis in parent cell clusters. Figs. 26A through 26C are violin plots showing gene set scores from gene set analysis in the four parent cell clusters. Each point lying on the violin plots in Figs. 26A through 26C represents a gene set score in a cell. In Fig. 26A, a higher gene set score for the gene set "upregulated in Tregs relative to downregulated in Teff" indicates an increase in the expression of the Treg cell phenotype, whereas a lower gene set score for the gene set "upregulated in Tregs relative to downregulated in Teff" indicates an increase in the expression of the Teff cell phenotype. In Fig. 26B, a higher gene set score for the "gradually activated upon differentiation into memory cells" gene set indicates an increase in the late memory T cell phenotype, whereas a lower gene set score for the "gradually activated upon differentiation into memory cells" gene set indicates an increase in the early memory T cell phenotype. In Fig. 26C, a higher gene set score for the "activated in TEM relative to suppressed in TN" gene set indicates an increase in the effector memory T cell phenotype, whereas a lower gene set score for the "activated in TEM relative to suppressed in TN" gene set indicates an increase in the naive memory T cell phenotype. Cells in cluster 3 are shown to be in the late, more differentiated memory T cell state compared to cells in cluster 1 and cluster 2, which are in the early, less differentiated memory T cell state. Cluster 0 appears to represent an intermediate state of T cells. Taken together, these data demonstrate that there is a significant level of heterogeneity among the cells of origin.

Fig. 27A, 27B and 27C. TCR sequencing and measurement of clonotype diversity. Day 9 cells had a flatter clonotype frequency distribution (higher diversity).

Fig. 28A and 28B . Flow cytometry analyses to determine CAR expression at days 4 and 7 post-transduction. Flow cytometry analyses of CAR T cells generated by co-transducing cells with vectors encoding BCMA and CD19 CARs at different MOI combinations using the ARM method in a 24-well plate. Fig. 28A . Flow cytometry plots showing the expression of BCMA mono-CAR, CD19 mono-CAR, and CAR in double+ cells at days 4 and 7 post-transduction under four different MOIs in addition to controls (UTD and single vector). Fig. 28B. Quantification of subsets within the CAR+ populations, including the total BCMA-CAR+ T cell population, the total CD19-CAR+ T cell population, and the total CAR+ T cell population (the sum of the two mono-CAR+ T cell groups and the two CAR+ T cell groups) under each condition, were determined as described in Fig. 28A. Data shown are representative of T cells from three donors with consistent results. CAR+ cell percentages are gated on the live CD3+ T cell population.

Fig. 29 . Flow cytometric analyses of CAR expression at day 4 post-transduction. Flow cytometric analyses of the final products of the dual-targeting CAR-T cocktail, mono-BCMA CAR-T, and mono-CD19 CAR-T for CAR expression at day 4 post-transduction. Small aliquots of each product collected at 24 hours were re-cultured for three days before flow cytometric staining.

Fig. 30A, 30B, 30C, 30D, and 30E . In vivo efficacy of dual-targeting CAR-T compared to mono-BCMA-CAR-T and CD19-CAR-T in xenograft models. NSG mice were injected with cell lines expressing a luciferase reporter gene (KMS-11, or Nalm-6, or a mixture of both with 5% Nalm-6-luc). Tumor burden is expressed as whole-body luminescence (photons/s) and depicted as mean tumor burden+SEM. On day 7 or 8 post-tumor inoculation, mice were treated with dual-targeting CAR-T cocktail, BCMA-CAR-T, or CD19-CAR-T at the appropriate doses (approximate number of viable CAR+ T cells). Vehicle medium (PBS) and untransduced T cells (UTD) served as negative controls. N=5 mice for all groups. BCMA-CAR-T and CD19-CAR-T served as respective controls at the highest dose level. All experiments were terminated at day 23 post-CAR-T administration.

Fig. 31 . Fig. 31 is a bar graph showing % mono-CD19-CAR+ cells, % mono-BCMA-CAR+ cells, and % double BCMA/CD19-CAR+ cells at day 4 post-transduction (day 3 post-harvest).

Fig. 32 . Characterization of T cell subsets. Fig. 32 is a graph showing the % of CD4+ T cells, CD8+ T cells, naive T cells (Tn), central memory T cells (Tcm), effector memory T cells (Tem), and effector memory T cells re-expressing CD45RA (TEMRA) in the starting material, enrichment material, and day 1 harvest material.

Fig. 33A and 33B . Kinetics of IFNγ in plasma of mice treated with BCMA/CD19 dual-specific CAR-T, BCMA-CAR-T, and CD19-CAR-T. Animals were treated with PBS, UTD, BCMA/CD19 dual-specific CAR-T, BCMA-CAR-T, or CD19-CAR-T at the corresponding CAR-T doses. Mice were bled and plasma cytokine levels were measured by MSD assay.

DETAILED DESCRIPTION

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The singular form refers to one or more (i.e. at least one) grammatical entities of the present application. As an example, "element" means one element or several elements.

It is intended that the term "approximately" with respect to a measurand such as a quantity, a time period, etc., covers deviations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as long as such deviations are acceptable for carrying out the disclosed methods.

The compositions and methods of the present invention encompass polypeptides and nucleic acids having the said sequences or sequences substantially identical or similar thereto, for example, sequences that are at least 85%, 90%, or 95% or more identical to said sequence. When used with respect to an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid sequence that comprises a sufficient or minimum number of amino acid residues that are i) identical to amino acid residues in a second amino acid sequence after alignment thereof or ii) are conservative substitutions thereof, such that the first and second amino acid sequences can share a common structural domain and/or have a common functional activity, for example, amino acid sequences that share a common structural domain are at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence, for example, the sequence presented in this document.

In relation to a nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that comprises a sufficient or minimum number of nucleotides that are identical to nucleotides in a second nucleic acid sequence when aligned such that the first and second nucleotide sequences encode polypeptides that share a common functional activity, or encode a common structural domain of the polypeptides or are responsible for a common functional activity of the polypeptides, e.g., the nucleotide sequences are characterized by at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence, for example, the sequence presented in this document.

The term "variant" refers to a polypeptide that has an amino acid sequence that is substantially identical to a reference amino acid sequence or is encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially identical to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and is capable of exhibiting one or more activities of the reference amino acid sequence.

The term "cytokine" (e.g., IL-2, IL-7, IL-15, IL-21, or IL-6) includes a full-length naturally occurring cytokine, a fragment, or a variant thereof, e.g., a functional variant (including fragments and functional variants thereof that have at least 10%, 30%, 50%, or 80% of the activity, e.g., immunomodulatory activity, of the naturally occurring cytokine). In some embodiments, the cytokine has an amino acid sequence that is substantially identical (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a naturally occurring cytokine or is encoded by a nucleotide sequence that is substantially identical (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a naturally occurring nucleotide sequence encoding a cytokine. In some embodiments, as is clear from the context, the cytokine further comprises a receptor domain, such as a domain that is a cytokine receptor (e.g., IL-15/IL-15R).

As used herein, the term "BCMA" refers to B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis factor receptor (TNFR) family and is predominantly expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligand is referred to as B-cell activator of the TNF family (BAFF) and proliferation-inducing ligand (APRIL). BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16, producing a 994-nucleotide primary mRNA transcript (NCBI accession no. NM_001192.2) that encodes a 184-amino acid protein (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described that may play a role in regulating BCMA expression (Laabi Y et al. Nucleic Acids Res. 1994; 22:1147-1154). Additional transcript variants of unknown significance have been described (Smirnova AS et al. Mol Immunol. 2008; 45(4):1179-1183). A second isoform, also known as TV4, has been identified (UniProt Identifier Q02223-2). As used herein, "BCMA" includes proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splice variants, of full-length wild-type BCMA.

The phrase "disease associated with BCMA expression" includes, but is not limited to, a disease associated with a cell that expresses BCMA (e.g., wild-type BCMA or mutant BCMA), or a condition associated with a cell that expresses BCMA (e.g., wild-type BCMA or mutant BCMA), including, for example, proliferative diseases such as cancer or malignancy, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancer indication associated with a cell that expresses BCMA (e.g., wild-type BCMA or mutant BCMA). For the avoidance of doubt, a disease associated with BCMA expression may include a condition associated with a cell that does not currently express BCMA, such as because BCMA expression has been suppressed, such as by treatment with a molecule that targets BCMA, such as a BCMA inhibitor described herein, but which at one time expressed BCMA. In one aspect, a cancer associated with BCMA expression (e.g., wild-type or mutant BCMA) is a hematological cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, a cancer associated with BCMA expression (e.g., wild-type or mutant BCMA) is a malignancy of differentiated plasma B cells. In one aspect, a cancer associated with the expression of BCMA (e.g., wild-type BCMA or mutant BCMA) includes forms of cancer and malignancy, including but not limited to, for example, one or more forms of acute leukemia, including but not limited to, for example, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL); one or more forms of chronic leukemia, including but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Additional cancers or hematologic conditions associated with BMCA expression (e.g., wild-type or mutant BCMA) include, but are not limited to, e.g., B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," which is a diverse group of hematological conditions united by ineffective formation (or dysplasia) of myeloid blood cells, etc. In some embodiments, the cancer is multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or glioblastoma. In some embodiments, the disease associated with BCMA expression includes a plasma cell proliferative disorder such as silent myeloma ("smoldering" multiple myeloma or smoldering myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, forms of plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloidosis in the form of light chain amyloidosis, and POEMS syndrome (also known as Crowe-Fukasa syndrome, Takatsuki disease, and PEP syndrome). Additional diseases associated with the expression of BCMA (e.g., wild-type BCMA or mutant BCMA) include, but are not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with the expression of BCMA (e.g., wild-type BCMA or mutant BCMA), e.g., cancers described herein, e.g., prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, or lung cancer.

Non-cancer conditions that are associated with BCMA (e.g., wild-type BCMA or mutant BCMA) include viral infections; e.g., those caused by HIV, fungal infections, e.g., those caused by C. neoformans ; autoimmune disease; e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris, and Sjogren's syndrome; inflammatory bowel disease, ulcerative colitis; allospecific immune response associated with transplantation, disorders associated with mucosal immunity; and adverse immune responses to biologics (e.g., factor VIII) in which humoral immunity is important. In some embodiments, a non-cancer indication associated with BCMA expression includes, but is not limited to, e.g., autoimmune disease (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, a cell expressing a tumor antigen expresses or has at some time expressed mRNA encoding a tumor antigen. In one embodiment, a cell expressing a tumor antigen produces a tumor antigen protein (e.g., wild-type or mutant), and wherein the tumor antigen protein may be present at normal levels or at reduced levels. In one embodiment, a cell expressing a tumor antigen produced a tumor antigen protein at detectable levels at one time, and then substantially did not produce a tumor antigen protein at detectable levels thereafter.

The term "chimeric antigen receptor" or alternatively "CAR" or "CAR molecule" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in a CAR polypeptide construct are on the same polypeptide chain, e.g., forming a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not contiguous with one another, e.g., are on different polypeptide chains, e.g., as provided in the RCAR described herein.

In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a CD3zeta primary signaling domain). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and/or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

A CAR comprising an antigen-binding domain (e.g., an scFv, a single-domain antibody, or a TCR (e.g., a TCR alpha chain binding domain or a TCR beta chain binding domain)) that targets a specific tumor marker X, where X may be a tumor marker described herein, is also referred to as an XCAR. For example, a CAR comprising an antigen-binding domain that targets BCMA is referred to as a CAR for BCMA. A CAR can be expressed in any cell, such as an immune effector cell described herein (e.g., a T cell or an NK cell).

The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity through specific signaling pathways, forming second messengers, or functioning as effectors by responding to such messengers.

As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multichain or single-chain or intact immunoglobulins, and may be obtained from natural sources or from recombinant sources. Antibodies may be tetrameric immunoglobulin molecules.

The term "antibody fragment" refers to at least one portion of an intact antibody or recombinant variants thereof and refers to an antigen-binding domain, e.g., an antigen-recognizing variable region of an intact antibody, sufficient to provide recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAbs (VL or VH), camelid VHH domains, and multispecific molecules formed from antibody fragments, such as a bivalent fragment comprising two or more, e.g., two, Fab fragments linked by a disulfide bridge at the hinge region, or two or more, e.g., two, linked isolated CDRs or other epitope-binding fragments of an antibody. The antibody fragment may also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments may also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide-based minibodies).

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are joined to each other in a contiguous position by a short flexible polypeptide linker and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, an scFv as used herein may have VL and VH variable regions arranged in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL. In some embodiments, the scFv may comprise the structure _NH2 -V _L -linker-V _H -COOH or NH2- _{V H} -linker-V _L -COOH.

The terms "complementarity determining region" or "CDR" as used herein refer to the amino acid sequences in the variable regions of an antibody that confer antigen specificity and binding affinity. For example, typically there are three CDRs (HCDR1, HCDR2, and HCDR3) in each variable region of the heavy chain, and three CDRs (LCDR1, LCDR2, and LCDR3) in each variable region of the light chain. The precise amino acid sequence boundaries of a given CDR may be determined using any of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (Chothia numbering scheme), or a combination thereof. In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.

The portion of the CAR composition of the present invention comprising the corresponding antibody or antibody fragment can exist in various forms, for example, in which the antigen-binding domain is expressed as part of a polypeptide chain, including, for example, as a fragment of a single-domain antibody (sdAb), a single-chain antibody (scFv), or, for example, a human or humanized antibody (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain in the CAR composition of the present invention comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment that comprises an scFv.

As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as a binding domain for a target) refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. The bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by the presence of a first sequence of the variable domain of the immunoglobulin, which is characterized by binding specificity to the first epitope, and a second sequence of the variable domain of the immunoglobulin, which is characterized by binding specificity to the second epitope.

The terms "bispecific antibody" and "bispecific antibodies" refer to molecules that combine the antigen-binding regions of two antibodies within a single molecule. Thus, a bispecific antibody is capable of binding two different antigens simultaneously or sequentially. Methods for producing bispecific antibodies are well known in the art. Various formats for combining two antibodies are also known in the art. Forms of bispecific antibodies of the present invention include, but are not limited to, diabody, single-chain diabody, Fab dimerization product (Fab-Fab), Fab-scFv, and tandem antibody, as known to those skilled in the art.

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, which generally defines the class to which the antibody belongs.

The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains are the two major isotypes of antibody light chains.

The term "recombinant antibody" refers to an antibody produced by recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be construed as meaning an antibody that has been produced by synthesizing a DNA molecule encoding an antibody, wherein the DNA molecule expresses a protein antibody or an amino acid sequence defining an antibody, wherein the DNA sequence or amino acid sequence has been obtained by recombinant DNA or amino acid sequencing technology available and well known in the art.

The term "antigen" or "Ag" refers to a molecule that elicits an immune response. Such an immune response may involve the production of antibodies or the activation of specific immune cells, or both. One skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Additionally, antigens may be derived from recombinant or genomic DNA. One skilled in the art will appreciate that any DNA that contains a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response thereby encodes an "antigen," as that term is used herein. Additionally, one skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is understood that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response. Moreover, one skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is understood that an antigen may be synthesized, or may be obtained from a biological sample, or may be a macromolecule other than a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a fluid with other biological components.

The terms "anti-tumor effect" and "anti-cancer effect" are used interchangeably and refer to a biological effect that can manifest itself in a variety of ways, including, but not limited to, for example, a reduction in tumor volume or cancer volume, a reduction in the number of tumor cells or cancer cells, a reduction in the number of metastases, an increase in life expectancy, a decrease in the level of tumor cell proliferation or cancer cell proliferation, a decrease in tumor cell survival or cancer cell survival, or an alleviation of various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" or "anti-cancer effect" can also manifest itself in the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the occurrence of a tumor or cancer in the first place.

The term "autologous" refers to any material obtained from the same individual into which it is later to be reintroduced.

The term "allogeneic" refers to any material obtained from another animal of the same species as the individual to which the material is administered. Two or more individuals are said to be allogeneic with respect to each other if their genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may have a degree of genetic diversity sufficient for them to interact antigenically.

The term "xenogeneic" refers to a transplant obtained from an animal of another species.

The term "apheresis" as used herein refers to an art-recognized extracorporeal technique in which blood from a donor or patient is withdrawn from the donor or patient and passed through a machine that separates selected specific components and returns the remainder to the donor or patient's bloodstream, such as by retransfusion. Thus, when referring to an "apheresis specimen," it refers to a specimen obtained using apheresis.

The term "cancer" refers to a disease characterized by rapid and uncontrolled growth of aberrant cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various forms of cancer are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. In some embodiments, cancers treated by the methods described herein include multiple myeloma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

The terms "tumor" and "cancer" are used interchangeably in this document, for example, both terms include solid tumors and tumors of liquid tissues, such as diffuse or circulating. As used in this document, the term "cancer" or "tumor" includes premalignant as well as malignant forms of cancer and tumors.

As used herein, the term "derived from" refers to a relationship between the first and second molecules. This generally refers to a structural similarity between the first molecule and the second molecule and does not have the additional meaning or include a method or source limitation with respect to the first molecule being derived from the second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3-zeta molecule, the intracellular signaling domain retains the CD3-zeta structure sufficiently to have the desired function, namely the ability to generate a signal under suitable conditions. This does not have the additional meaning or include a limitation with respect to a particular method of obtaining the intracellular signaling domain, for example, it does not mean that in order to have an intracellular signaling domain, one must start with a CD3-zeta sequence and remove unwanted sequence or introduce mutations to obtain the intracellular signaling domain.

The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody or antibody fragment comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

The term "stimulation" when applied to stimulation by a stimulatory and/or costimulatory molecule refers to a response, such as a primary or secondary response, induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) and/or a costimulatory molecule (e.g., CD28 or 4-1BB) to its cognate ligand that mediates a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate a change in the expression of certain molecules and/or a reorganization of cytoskeletal structures, etc.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides primary cytoplasmic signaling sequence(s) that regulates primary activation of the TCR complex in a stimulatory manner in at least some aspects of the T cell signaling pathway. In some embodiments, the ITAM-containing domain in a CAR recapitulates primary TCR signaling independent of endogenous TCR complexes. In some embodiments, the primary signal is initiated, for example, by binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, and this results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signal sequence (also referred to as the "primary signaling domain"), which acts in a stimulatory manner, may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM-containing primary cytoplasmic signal sequence that is particularly useful in the present invention include, but are not limited to, sequences derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD66d, DAP10 and DAP12. In a particular CAR of the present invention, the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, such as a CD3 zeta primary signaling sequence. The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as a helper cell (e.g., B cell, dendritic cell, etc.), that presents a foreign antigen complexed with major histocompatibility complex (MHC) molecules on its surface. T cells can recognize these complexes through their T cell receptors (TCRs). APCs process the antigens and present them to T cells.

As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. In some embodiments, the intracellular signaling domain transmits a signal to an effector function and directs the cell to perform a specialized function. Although the entire intracellular signaling domain may be used, in many cases it is not necessary to use the entire chain. In the event that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact chain, so long as it transmits a signal to an effector function. Thus, the term "intracellular signaling domain" is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit a signal to an effector function.

The intracellular signaling domain generates a signal that promotes immune effector function by a cell containing a CAR, such as a CAR-T cell. Examples of immune effector function, such as in a CAR-T cell, include cytolytic activity and helper activity, including secretion of cytokines.

In some embodiments, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include domains derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include domains derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence of a coreceptor or costimulatory molecule.

The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, sequences derived from CD3-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12.

The term "zeta" or alternatively "zeta chain", "CD3 zeta" or "TCR zeta" refers to CD247. Swiss-Prot Accession Number P20963 provides exemplary amino acid sequences of human CD3 zeta. "Zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" refers to a CD3 zeta stimulatory domain or a variant thereof (e.g., a molecule having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, the cytoplasmic zeta domain comprises residues 52-164 of the sequence of GenBank Accession Number BAG36664.1 or a variant thereof (e.g., a molecule having mutations, e.g., point mutations, fragments, insertions, or deletions). In some embodiments, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence set forth in SEQ ID NO: 9 or 10, or a variant thereof (e.g., a molecule having mutations, such as point mutations, fragments, insertions, or deletions).

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory T cell response such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are essential for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecule, TNF protein receptors, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activating receptors, BTLA, Toll-like receptor ligand, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD28-OX40, CD28-4-1BB and a ligand that specifically binds to CD83.

The costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

The intracellular signaling domain may comprise the entire intracellular portion or the entire native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

The term "4-1BB" refers to CD137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot accession number P20963 provides exemplary amino acid sequences of human 4-1BB. A "4-1BB costimulatory domain" refers to a 4-1BB costimulatory domain or a variant thereof (e.g., a molecule having mutations, such as point mutations, fragments, insertions, or deletions). In some embodiments, the "4-1BB costimulatory domain" is the sequence set forth in SEQ ID NO: 7 or a variant thereof (e.g., a molecule having mutations, such as point mutations, fragments, insertions, or deletions).

As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response, such as by facilitating an immune effector response. Examples of immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

As used herein, the term "immune effector function or immune effector response" refers to a function or response, such as that of an immune effector cell, that enhances or facilitates an immune attack on a target cell. For example, an immune effector function or response refers to a property of a T or NK cell that facilitates the killing or inhibition of the growth or proliferation of a target cell. In the case of a T cell, priming and costimulation are examples of immune effector functions or responses.

The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

The term "coding" refers to the intrinsic property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis, in biological processes, of other polymers and macromolecules having the specified nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or the specified amino acid sequence and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein if the protein is produced by transcription and translation of the mRNA corresponding to the gene in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is commonly represented in sequence listings, and the noncoding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of the gene or cDNA.

Unless otherwise specified, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence. The phrase "a nucleotide sequence that encodes a protein or RNA" may also include introns to the extent that a nucleotide sequence encoding a protein may, in some variant, contain an intron(s).

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, composition, material, or formulation described herein effective to achieve a particular biological result.

The term "endogenous" refers to any material derived from or produced within an organism, cell, tissue, or system.

The term "exogenous" refers to any material introduced into or produced outside an organism, cell, tissue, or system.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence. In some embodiments, expression includes translation of mRNA introduced into a cell.

The term "transfer vector" refers to a composition that contains an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or virus. This term should also be interpreted in such a way that it additionally includes non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, etc. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

The term "expression vector" refers to a vector that contains a recombinant polynucleotide containing expression control sequences operably linked to a nucleotide sequence to be expressed. The expression vector contains a sufficient number of cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) in which a recombinant polynucleotide is incorporated.

The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, so as vectors they are one of the most efficient ways to deliver genes. HIV, SIV, and FIV are all examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, but not limited to, the self-inactivating lentiviral vector disclosed in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical practice include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, and the like. Non-clinical types of lentiviral vectors are also available and should be known to one of skill in the art.

The term "homologous" or "identity" refers to the identity of the subunit sequences of two polymer molecules, for example, two nucleic acid molecules such as two DNA molecules or two RNA molecules, or two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomeric subunit, for example, if a position in each of two DNA molecules is occupied by an adenine, then they are homologous or identical at that position. The homology between two sequences is directly proportional to the number of positions that match or are homologous; for example, if half (e.g., five positions in a ten-subunit polymer) of the positions in two sequences are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies and fragments of these antibodies are human immunoglobulins (the recipient antibody or antibody fragment) in which residues from the complementarity determining region (CDR) of the recipient have been replaced by residues from the CDR of a non-human species (the donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, residues of the framework region (FR) of the human Fv immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody/antibody fragment may comprise residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications can further improve and optimize the functional characteristics of the antibody or antibody fragment. Typically, a humanized antibody or antibody fragment will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin and all or a substantial portion of the FR regions are regions of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of the constant region (Fc) of an immunoglobulin, typically a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

"Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

The term "isolated" means altered from or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from materials accompanying it in its natural state is "isolated." An isolated nucleic acid or protein may be in a substantially purified form or may be in a non-native environment, such as a host cell.

In the context of the present invention, the following abbreviations are used for common nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The term "operably linked" or "transcriptional control" refers to a functional relationship between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. The operably linked DNA sequences may be contiguous with each other and, for example, if it is necessary to connect two protein coding regions that are in the same reading frame.

The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral administration, or infusion techniques.

The term "nucleic acid", "nucleic acid molecule", "polynucleotide" or "polynucleotide molecule" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to those of the reference nucleic acid and are metabolized by a mechanism similar to that of naturally occurring nucleotides. In some embodiments, a "nucleic acid", "nucleic acid molecule", "polynucleotide" or "polynucleotide molecule" provides a nucleotide/nucleoside derivative or analog. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its variants with conservative modifications (e.g., with degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence specified explicitly. In particular, degenerate codon substitutions, e.g., conservative substitutions, can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with any of the canonical bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, but not limited to, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. The polypeptide includes a natural peptide, a recombinant peptide, or a combination of both.

The term "promoter" refers to a DNA sequence recognized by the cellular or introduced nucleotide apparatus that is required to initiate specific transcription of a polynucleotide sequence.

The term "promoter/regulatory sequence" refers to a nucleic acid sequence that is necessary for the expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, the sequence may also contain an enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. For example, a promoter/regulatory sequence may be a sequence that ensures the expression of a gene product in a tissue-specific manner.

The term "constitutive promoter" refers to a nucleotide sequence that, when in functional association with a polynucleotide that codes for or specifies a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

The term "inducible promoter" refers to a nucleotide sequence that, when in functional association with a polynucleotide that codes for or specifies a gene product, causes the production of the gene product in a cell substantially only when an inducer that corresponds to the promoter is present in the cell.

The term "tissue-specific promoter" refers to a nucleotide sequence that, when in functional association with a polynucleotide that encodes or specifies a gene, causes the production of the gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

The terms "cancer-associated antigen," "tumor antigen," "hyperproliferative disorder-associated antigen," and "hyperproliferative disorder-associated antigen" interchangeably refer to antigens that are common to specific hyperproliferative disorders. In some embodiments, such terms refer to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of a cancer cell in whole or as a fragment (e.g., an MHC molecule/peptide) and that is useful for preferentially targeting a pharmacologic agent to a cancer cell. In some embodiments, the tumor antigen is a marker expressed by both normal cells and cancer cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in a cancer cell compared to a normal cell, such as 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more than a normal cell. In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized in a cancer cell, such as a molecule that contains deletions, additions, or mutations compared to the molecule expressed on a normal cell. In some embodiments, the tumor antigen will be expressed exclusively on the cell surface of a cancer cell, in whole or in part (e.g., an MHC molecule/peptide), and will not be synthesized or expressed on the surface of a normal cell. In some embodiments, the hyperproliferative disorder-associated antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, forms of leukemia, uterine cancer, cervical cancer, bladder cancer, renal cancer, and forms of adenocarcinoma such as breast cancer, prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), ovarian cancer, pancreatic cancer, and the like, or a plasma cell proliferative disorder such as asymptomatic myeloma ("smoldering" multiple myeloma or smoldering myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, forms of plasmacytomas (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloidosis in the form of light chain amyloidosis, and POEMS syndrome (also known as Crowe-Fukasa syndrome, Takatsuki disease, and PEP syndrome). In some embodiments, CARs of the present invention include CARs that comprise an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to a peptide presented by an MHC molecule. Typically, peptides derived from endogenous proteins fill pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I molecule complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and/or tumor-specific peptide/MHC molecule complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the human leukocyte antigen (HLA)-A1 or HLA-A2 frame have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935–1942; Sergeeva et al., Blood, 2011 117(16):4262–4272; Verma et al., J Immunol 2010 184(4):2156–2165; Willemsen et al., Gene Ther 2001 8(21):1601–1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84–100). For example, a TCR-like antibody can be identified by screening a library such as a human scFv phage display library.

The terms "tumor maintenance antigen" or "cancer maintenance antigen" refer interchangeably to a molecule (usually a protein, carbohydrate, or lipid) that is expressed on the surface of a cell that is not itself cancerous but that provides maintenance to cancer cells, such as by promoting their growth or survival, such as resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). A tumor maintenance antigen does not necessarily itself play a role in the maintenance of tumor cells, so long as the antigen is present in a cell that provides maintenance to cancer cells.

The term "flexible polypeptide linker" or "linker" as used in connection with scFv refers to a peptide linker that is comprised of amino acids, such as glycine and/or serine residues, used alone or in combination to link a heavy chain variable region and a light chain variable region to each other. In some embodiments, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, wherein n is a positive integer that is equal to or greater than 1 (SEQ ID NO: 41). For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In some embodiments, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4 Ser)3 (SEQ ID NO: 28). In some embodiments, linkers comprise multiple repeats of (Gly2Ser) (GlySer) or (Gly3Ser) (SEQ ID NO: 29). Also included within the scope of the present invention are the linkers described in WO2012/138475, incorporated herein by reference.

As used herein, the 5' cap (also called the RNA cap, 7-methylguanosine RNA cap, or m7G RNA cap) is a modified guanine nucleotide that is added to the "front," or 5' end, of eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group that is linked to the first nucleotide transcribed. Its presence is critical for ribosome recognition and protection from RNases. Cap addition is coupled to transcription and occurs cooperatively with transcription, such that each event influences the other. Shortly after transcription begins, the 5' end of nascent mRNA binds to the RNA polymerase-associated cap synthesizing complex. This enzymatic complex catalyzes the chemical reactions required to cap the mRNA. Synthesis occurs as a multistep biochemical reaction. The capping moiety can be modified to modulate functional properties of the mRNA, such as its stability or translation efficiency.

As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. In some embodiments, the RNA is mRNA. Typically, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains a template used to produce in vitro transcribed RNA.

As used herein, "poly(A)" is a consecutive series of adenosine residues attached by polyadenylation to an mRNA. In some embodiments of a transient expression construct, the poly(A) length is from 50 to 5000 (SEQ ID NO: 30). In some embodiments, the poly(A) length is greater than 64. In some embodiments, the poly(A) length is greater than 100. In some embodiments, the poly(A) length is greater than 300. In some embodiments, the poly(A) length is greater than 400. Poly(A) sequences can be modified by chemical means or enzymes to modulate functional properties of mRNA, such as localization, stability, or translation efficiency.

As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or a modified version thereof to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA by the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and its associated protein help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but may also occur later in the cytoplasm. Following transcription termination, the mRNA strand is cleaved by an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

As used herein, "transient" refers to the expression of a non-integrated transgene over a period of hours, days, or weeks, where the expression time period is shorter than the time period for expression of the gene if it is integrated into the genome or contained in a stable plasmid replicon in a host cell.

As used herein, the terms "treat," "treating," and "treating" refer to reducing or ameliorating the progression, severity, and/or duration of a proliferative disorder or alleviating one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder by administering one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the present invention). In particular embodiments, the terms "treat," "treating," and "treating" refer to alleviating at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible to a patient. In other embodiments, the terms "treat," "treating," and "treating" refer to inhibiting the progression of a proliferative disorder, either at a physical level, such as by stabilizing a discernible symptom, or at a physiological level, such as by stabilizing a physical parameter, or both. In other embodiments, the terms "treat," "treating," and "treating" refer to reducing or stabilizing the size of a tumor or the number of cancer cells.

The term "signal transduction pathway" refers to the biochemical relationship between the various signal carrier molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that are capable of receiving a signal and transmitting a signal across the cell membrane.

The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g. mammals, humans).

The term "substantially purified cell" refers to a cell that is substantially free of other cell types. A "substantially purified cell" also refers to a cell that has been separated from other cell types with which it is normally associated in its naturally occurring state. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that has been separated from the cells with which it is naturally associated in its natural state. In some embodiments, the cells are cultured in vitro. In some embodiments, the cells are not cultured in vitro.

As used in this document, the term "therapeutic" means treatment. The therapeutic effect is obtained by alleviating, suppressing, remitting, or eliminating a disease state.

As used in this document, the term "prophylaxis" means the prevention or prophylactic treatment of a disease or condition.

The terms "transfected" or "transformed" or "transduced" refer to a process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such a cell includes the primary cell of the subject and its progeny.

The term "specifically binds" refers to an antibody or ligand that recognizes and binds its cognate protein binding partner (e.g., a stimulatory and/or costimulatory molecule present on a T cell) present in a sample, but the antibody or ligand does not substantially recognize or bind other molecules in the sample.

As used herein, a "regulated chimeric antigen receptor (RCAR)" refers to a collection of polypeptides, typically two in the simplest embodiments, that when located in an immune effector cell, confers on the cell specificity for a target cell, typically a cancer cell, and the generation of an intracellular signal. In some embodiments, an RCAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or a costimulatory molecule as defined herein with respect to a CAR molecule. In some embodiments, the polypeptides in a collection of an RCAR are not contiguous with one another, such as being on different polypeptide chains. In some embodiments, the RCAR comprises a dimerization switch that, when present, can link the polypeptides to each other, such as can link an antigen-binding domain to an intracellular signaling domain. In some embodiments, the RCAR is expressed in a cell (e.g., an immune effector cell) described herein, such as an RCAR-expressing cell (also referred to herein as an "RCARX cell"). In some embodiments, the RCARX cell is a T cell and is referred to as an RCAR-T cell. In some embodiments, the RCARX cell is an NK cell and is referred to as an RCAR-N cell. The RCAR can confer on the RCAR-expressing cell specificity for a target cell, typically a cancer cell, and regulated intracellular signal generation or proliferation, which can optimize the immune effector property of the RCAR-expressing cell. In some embodiments, the RCAR cell is at least partially dependent on the antigen-binding domain to provide specificity for a target cell that comprises an antigen bound by the antigen-binding domain.

As used herein, the term "membrane anchor" or "membrane attachment domain" refers to a polypeptide or moiety, such as a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

As used herein, the term "switch domain," for example, in relation to RCAR, refers to an entity, typically a polypeptide entity, that associates with another switch domain in the presence of a dimerizing molecule. The association results in operative linkage of a first entity linked, for example, fused, to the first switch domain, and a second entity linked, for example, fused, to the second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In some embodiments, the first and second switch domains are the same, for example, they are polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In some embodiments, the first and second switch domains are different from each other, for example, they are polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerization switch. In some embodiments, the switch is intracellular. In some embodiments, the switch is extracellular. In some embodiments, the switch domain is a polypeptide entity, such as an FKBP or FRB-based entity, and the dimerizing molecule is a small molecule, such as a rapalog. In some embodiments, the switch domain is a polypeptide entity, such as an scFv, that binds a myc peptide, and the dimerizing molecule is a polypeptide, a fragment thereof, or a polypeptide multimer, such as a myc ligand or myc ligand multimers that bind to one or more scFvs for myc. In some embodiments, the switch domain is a polypeptide entity, such as a receptor for myc, and the dimerizing molecule is an antibody or fragments thereof, such as an anti-myc antibody.

As used herein, the term "dimerizing molecule," for example, in relation to RCAR, refers to a molecule that facilitates the association of a first switch domain with a second switch domain. In some embodiments, the dimerizing molecule does not naturally occur in the subject or does not occur at concentrations that can result in a significant degree of dimerization. In some embodiments, the dimerizing molecule is a small molecule, such as rapamycin or a rapalog, such as RAD001.

The term "low immune response enhancing dose" when used in relation to an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of the mTOR inhibitor that provides partial, but not complete, inhibition of mTOR activity, such as as measured by inhibition of P70-S6 kinase activity. Methods for assessing mTOR activity, such as by inhibition of P70-S6 kinase, are discussed herein. The dose is insufficient to cause complete immune suppression, but sufficient to enhance the immune response. In some embodiments, a low immune response enhancing dose of the mTOR inhibitor results in a decrease in PD-1 positive T cells and/or an increase in PD-1 negative T cells or an increase in the PD-1 negative T cell/PD-1 positive T cell ratio. In some embodiments, a low dose of an immune response enhancing mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, a low dose of an immune response enhancing mTOR inhibitor results in one or more of the following:

increased expression of one or more of the following markers: CD62L ^high , CD127 ^high , CD27 ⁺ , and BCL2, e.g. on memory T cells, e.g. on memory T cell precursors;

decreased expression of KLRG1, such as on memory T cells, such as on memory T cell precursors; and

increasing the number of memory T cell precursors, such as cells with any or a combination of the following characteristics: elevated CD62L ^high , elevated CD127 ^high , elevated CD27 ⁺ , decreased KLRG1, and elevated BCL2;

where any of the changes described above occurs, for example, at least transiently, for example, compared to a subject not receiving treatment.

As used herein, "refractory" refers to a disease, such as cancer, that does not respond to treatment. In some embodiments, refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, refractory cancer may become resistant during treatment. Refractory cancer is also referred to as resistant cancer.

As used herein, "recurrent" or "relapse" refers to the return or reappearance of a disease (e.g., cancer) or the signs and symptoms of a disease, such as cancer, after a period of improvement or responsiveness, such as following prior treatment with a therapy, such as an anti-cancer therapy. An initial responsiveness period may include a drop in cancer cell levels below a certain threshold, such as below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may include an increase in cancer cell levels above a certain threshold, such as above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance may include, for example, the reappearance of blast cells in the blood, bone marrow (> 5%), or any extramedullary site after a complete response has been achieved. A complete response in this context may include <5% blast cells in the BM. More broadly, in some embodiments, a response (e.g., a complete response or a partial response) may include no detectable MRD (minimal residual disease). In some embodiments, the initial period of susceptibility lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

Ranges: Throughout this disclosure, various embodiments of the present invention may be presented in range format. It should be understood that a description in range format is for convenience and brevity only and should not be considered as an inflexible limitation on the scope of the present invention. Accordingly, a description of a range should be considered as specifically disclosing all possible subranges, as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose such sub-ranges as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes anything with 95%, 96%, 97%, 98%, or 99% identity and includes such sub-ranges as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

As used herein, the term "gene editing system" refers to a system, such as one or more molecules, that direct and effect a change, such as a deletion, in one or more nucleic acids at or near a site in genomic DNA that is targeted by the system. Gene editing systems are known in the art and are described in more detail below.

As used herein, administered "in combination" means that two (or more) different treatments are delivered to a subject during a period when the subject is suffering from a disorder, such as two or more treatments being delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has been discontinued for other reasons. In some embodiments, delivery of one treatment is still occurring when delivery of the second begins, such that there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "parallel" delivery. In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments, in either case, the treatment is more effective due to the combined administration. For example, the second treatment is more effective, such that an equivalent effect is observed with a smaller amount of the second treatment, or the second treatment reduces the intensity of symptoms to a greater extent than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation is observed with the first treatment. In some embodiments, the delivery is such that the reduction in the intensity of a symptom or other parameter associated with the disorder is greater than would be observed with the delivery of one treatment in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may be such that the effect of the first treatment delivered is still detectable with the delivery of the second.

The terms "depletion" or "deplete" used interchangeably herein refer to a decrease or reduction in the level or amount of a cell, protein, or macromolecule in a sample after a process, such as a selection step, such as negative selection, has been performed. The depletion may be a complete or partial depletion of the cell, protein, or macromolecule. In some embodiments, the depletion is at least a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease or reduction in the level or amount of the cell, protein, or macromolecule compared to the level or amount of the cell, protein, or macromolecule in the sample before the process has been performed.

As used herein, a "naive T cell" refers to a T cell that is antigen-naive. In some embodiments, a T cell that is antigen-naive has encountered its cognate antigen in the thymus but not in the periphery. In some embodiments, naive T cells are precursors to memory cells. In some embodiments, naive T cells express both CD45RA and CCR7 but do not express CD45RO. In some embodiments, naive T cells may be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127 and lack CD95 or the CD45RO isoform. In some embodiments, the naive T cells express CD62L, IL-7 receptor α, IL-6 receptor, and CD132, but do not express CD25, CD44, CD69, or CD45RO. In some embodiments, the naive T cells express CD45RA, CCR7, and CD62L, and do not express CD95 or IL-2 receptor β. In some embodiments, surface marker expression levels are assessed by flow cytometry.

The term "central memory T cells" refers to a subset of T cells that in humans are CD45RO positive and express CCR7. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R, and/or IL-15R. In some embodiments, central memory T cells express CD45RO, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, surface marker expression levels are assessed by flow cytometry.

The term "memory T stem cells", "memory T cells that are stem cells", "memory T cells that are like stem cells", "stem T cells that are memory cells", "stem cells that are memory T cells", "memory cells that are stem T cells" or "TSCM cells" refers to a subset of memory T cells with stem cell-like capabilities, such as self-renewal capabilities and/or multipotent potential to convert to memory T cell and/or effector T cell subsets. In some embodiments, the memory T stem cells express CD45RA, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, surface marker expression levels are assessed by flow cytometry. In some embodiments, exemplary memory T stem cells are disclosed in Gattinoni et al., Nat Med. 2017 January 06; 23(1): 18-27, incorporated herein by reference in its entirety.

For clarity, unless otherwise indicated, classification of a cell or population of cells as "non-expressing" or "absent" or "negative for" a particular marker may not necessarily mean the absolute absence of the marker. One of skill in the art can readily compare a cell to a positive and/or negative control and/or establish a predetermined threshold and classify a cell or population of cells as non-expressing or negative for the marker if the cell has an expression level below the predetermined threshold or the population of cells has an overall expression level below the predetermined threshold, using conventional detection methods, such as flow cytometry, such as described in the examples herein.

As used herein, the term "gene set score (upregulated in TEM relative to downregulated in TSCM)" for a cell refers to a score that reflects the degree to which the cell exhibits an effector memory T cell (TEM) phenotype compared to a stem cell memory T cell (TSCM) phenotype. A higher gene set score (upregulated in TEM relative to downregulated in TSCM) indicates an increase in the expression of the TEM phenotype, whereas a lower gene set score (upregulated in TEM relative to downregulated in TSCM) indicates an increase in the expression of the TSCM phenotype. In some embodiments, the gene set score (upregulated in TEMs versus downregulated in TSCMs) is determined by measuring the expression of one or more genes that are upregulated in TEM cells and/or downregulated in TSCM cells, such as one or more genes selected from the group consisting of MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RAB27B, ERN1, NPC1, NBEAL2, APOBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PLEKHA5, ADRB2, PLXND1, GNAO1, THBS1, PPP2R2B, CYTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY, and GFPT2. In some embodiments, the gene set score (upregulated in TEMs relative to downregulated in TSCM) is determined for each cell using RNA sequencing, such as single cell RNA sequencing (scRNA-seq), as illustrated, for example, in Example 7 with respect to Fig. 25A. In some embodiments, the gene set score (upregulated in TEMs relative to downregulated in TSCM) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used herein, the term "gene set score (up-regulated in Tregs versus down-regulated in Teff)" for a cell refers to a score that reflects the degree to which the cell exhibits a regulatory T cell (Treg) phenotype compared to an effector T cell (Teff) phenotype. A higher gene set score (up-regulated in Tregs versus down-regulated in Teff) indicates an increase in the expression of the Treg phenotype, whereas a lower gene set score (up-regulated in Tregs versus down-regulated in Teff) indicates an increase in the expression of the Teff phenotype. In some embodiments, the gene set score (upregulated in Tregs versus downregulated in Teffs) is determined by measuring the expression of one or more genes that are upregulated in Tregs and/or downregulated in Teffs, such as one or more genes selected from the group consisting of C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DPB2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf53, LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA-DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, CCR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2, LGALS1, ANXA2, PI16, DUSP4, LAYN, ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, LOC541471, RASGRP4, BCAS1, UTS2, MIAT, PRDM1, SEMA3G, FAM129A, HPGD, NCF4, LGALS3, CEACAM4, JAKMIP1, TIGIT, HLA-DRA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIB1, FCRL3, and FOXP3. In some embodiments, the gene set score (upregulated in Tregs versus downregulated in Teff) is determined using RNA sequencing, such as single cell RNA sequencing (scRNA-seq), as illustrated, for example, in Example 7 with respect to Fig. 25B. In some embodiments, the gene set score (upregulated in Tregs versus downregulated in Teff) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used herein, the term "stemness down-regulated gene set score" for a cell refers to a score that reflects the extent to which the cell exhibits a stemness phenotype. A lower stemness down-regulated gene set score indicates an increase in the expression of the stemness phenotype. In some embodiments, the stemness down-regulated gene set score is determined by measuring the expression of one or more genes that are up-regulated in a differentiating stem cell relative to genes that are down-regulated in a hematopoietic stem cell, such as one or more genes selected from the group consisting of ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, MEOX1, SFRP1, SP7, SRF, TAL1, and XRCC5. In some embodiments, the score of a gene set (repressed in stemness) is determined using RNA sequencing, such as single cell RNA sequencing (scRNA-seq), such as illustrated in Example 7 with respect to Fig. 25C. In some embodiments, the score of a gene set (repressed in stemness) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used in this document, the term "hypoxia-activated gene set score" for a cell refers to a score that reflects the degree to which the cell exhibits a hypoxia-specific phenotype. A higher hypoxia-activated gene set score indicates an increase in the expression of the hypoxia-specific phenotype. In some embodiments, the gene set score (upregulated in hypoxia) is determined by measuring the expression of one or more genes that are upregulated in cells exposed to hypoxia, such as one or more genes selected from the group consisting of ABCB1, ACAT1, ADM, ADORA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA, ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, BNIP3L, BPI, BTG1, C11orf2, C7orf68, CA12, CA9, CALD1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, CITED2, CLK1, CNOT7, COL4A5, COL5A1, COL5A2, COL5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1-Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, EIF4A3, ELF3, ELL2, ENG, ENO1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HMOX1, HMOX2, HSPA5, HSPD1, HSPH1, HYOU1, ICAM1, ID2, IFI27, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IL6, IL8, INSIG1, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LGALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13, MMP2, MMP7, MPI, MT1L, MTL3P, MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2, NOS2P1, NOS2P2, NOS3, NR3C1, NR4A1, NT5E, ODC1, P4HA1, P4HA2, PAICS, PDGFB, PDK3, PFKFB1, PFKFB3, PFKFB4, PFKL, PGAM1, PGF, PGK1, PGK2, PGM1, PIM1, PIM2, PKM2, PLAU, PLAUR, PLIN2, PLOD2, PNN, PNP, POLM, PPARA, PPAT, PROK1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RIOK3, RNASEL, RPL36A, RRP9, SAT1, SERPINB2, SERPINE1, SGSM2, SIAH2, SIN3A, SIRPA, SLC16A1, SLC16A2, SLC20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A16, SLC6A6, SLC6A8, SORL1, SPP1, SRSF6, SSSCA1, STC2, STRA13, SYT7, TBPL1, TCEAL1, TEK, TF, TFF3, TFRC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, THBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC, VIM, VPS11 and XRCC6. In some embodiments, the gene set score (upregulated in hypoxia) is determined using RNA sequencing, such as single cell RNA sequencing (scRNA-seq), such as illustrated in Example 7 with respect to Fig. 25D. In some embodiments, the gene set score (upregulated in hypoxia) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used herein, the term "autophagy-activated gene set score" for a cell refers to a score that reflects the degree to which the cell exhibits an autophagy-promoting phenotype. A higher autophagy-promoting gene set score indicates an increase in the expression of the autophagy-promoting phenotype. In some embodiments, the gene set score (activated during autophagy) is determined by measuring the expression of one or more genes that are activated in cells undergoing autophagy, such as one or more genes selected from the group consisting of ABL1, ACBD5, ACIN1, ACTRT1, ADAMTS7, AKR1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB, ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, ATG16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, C11orf2, C11orf41, C12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CALCOCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, CISD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRAM2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48A, FANCC, FANCF, FANCL, FBXO7, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP1L, FOXO1, FUNDC1, FUNDC2, FXR2, GABARAP, GABARAPL1, GABARAPL2, GABARAPL3, GABRA5, GDF5, GMIP, HAP1, HAPLN1, HBXIP, HCAR1, HDAC6, HGS, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HK2, HMGB1, HPR, HSF2BP, HSP90AA1, HSPA8, IFI16, IPPK, IRGM, IST1, ITGB4, ITPKC, KCNK3, KCNQ1, KIAA0226, KIAA1324, KRCC1, KRT15, KRT73, LAMP1, LAMP2, LAMTOR1, LAMTOR2, LAMTOR3, LARP1B, LENG9, LGALS8, LIX1, LIX1L, LMCD1, LRRK2, LRSAM1, LSM4, MAP1A, MAP1LC3A, MAP1LC3B, MAP1LC3B2, MAP1LC3C, MAP1S, MAP2K1, MAP3K12, MARK2, MBD5, MDH1, MEX3C, MFN1, MFN2, MLST8, MRPS10, MRPS2, MSTN, MTERFD1, MTMR14, MTMR3, MTOR, MTSS1, MYH11, MYLK, MYOM1, NBR1, NDUFB9, NEFM, NHLRC1, NME2, NPC1, NR2C2, NRBF2, NTHL1, NUP93, OBSCN, OPTN, P2RX5, PACS2, PARK2, PARK7, PDK1, PDK4, PEX13, PEX3, PFKP, PGK2, PHF23, PHYHIP, PI4K2A, PIK3C3, PIK3CA, PIK3CB, PIK3R4, PINK1, PLEKHM1, PLOD2, PNPO, PPARGC1A, PPY, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PRKD2, PRKG1, PSEN1, PTPN22, RAB12, RAB1A, RAB1B, RAB23, RAB24, RAB33B, RAB39, RAB7A, RB1CC1, RBM18, REEP2, REP15, RFWD3, RGS19, RHEB, RIMS3, RNF185, RNF41, RPS27A, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, S100A8, S100A9, SCN1A, SERPINB10, SESN2, SFRP4, SH3GLB1, SIRT2, SLC1A3, SLC1A4, SLC22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, SLC6A1, SLCO1A2, SMURF1, SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, SPATA18, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, STK11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOMM7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VPS33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593, and ZNF681. In some embodiments, the gene set score (activated during autophagy) is determined by RNA sequencing, such as single cell RNA sequencing (scRNA-seq), such as illustrated in Example 7 with respect to Fig. 25E. In some embodiments, the gene set score (activated by autophagy) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used herein, the term "gene set score (activated in the resting state relative to repressed in the activated state)" for a cell refers to a score that reflects the degree to which the cell exhibits a resting T cell phenotype compared to an activated T cell phenotype. A higher gene set score (activated in the resting state relative to repressed in the activated state) indicates an increase in the expression of the resting T cell phenotype, whereas a lower gene set score (activated in the resting state relative to repressed in the activated state) indicates an increase in the expression of the activated T cell phenotype. In some embodiments, the gene set score (upregulated in the resting state versus downregulated in the activated state) is determined by measuring the expression of one or more genes that are upregulated in resting T cells and/or downregulated in activated T cells, such as one or more genes selected from the group consisting of ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, CD248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, LZTFL1, MANBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISCH, NKTR, NLRP1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1, PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR, PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, PXN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF38, RNF44, ROR1, RPL30, RPL32, RPLP1, RPS20, RPS24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4C, SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG, SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SNPH, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, TECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM66, TMEM8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2, USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBED5, ZBTB25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZFP36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZNF350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862, and ZNF91. In some embodiments, the gene set score (upregulated in the quiescent state versus downregulated in the activated state) is determined by RNA sequencing, such as single cell RNA sequencing (scRNA-seq), such as illustrated in Example 7 with respect to Fig. 24D. In some embodiments, the gene set score (activated in the quiescent state relative to repressed in the activated state) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used in this document, the term "gene set (progressively upregulated upon differentiation into memory cells) score" for a cell refers to a score that reflects the stage of differentiation into memory cells. A higher gene set (progressively upregulated upon differentiation into memory cells) score indicates an increase in the expression of the late memory T cell phenotype, whereas a lower gene set (progressively upregulated upon differentiation into memory cells) score indicates an increase in the expression of the early memory T cell phenotype. In some embodiments, the gene set score (activated during autophagy) is determined by measuring the expression of one or more genes that are activated during differentiation into memory cells, such as one or more genes selected from the group consisting of MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, C11orf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYO5A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMIP, NAT13, TGFB1, FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3, ADCY9, CD300A, SCD5, ABI3, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, CLIC1, EIF2C4, MAP3K5, IL2RB, PLEKHG1, MYO6, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3, TP53INP1, MBOAT1, GYG1, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, and KLRB1. In some embodiments, the gene set score (gradually upregulated upon differentiation into memory cells) is determined using RNA sequencing, such as single cell RNA sequencing (scRNA-seq), as illustrated, for example, in Example 7 with respect to Fig. 26B. In some embodiments, the gene set score (gradually upregulated upon differentiation into memory cells) is calculated using the average log-normalized gene expression value for all genes in the gene set.

As used herein, the term "gene set score (upregulated in TEM relative to downregulated in TN)" for a cell refers to a score that reflects the degree to which the cell exhibits an effector memory T cell (TEM) phenotype compared to a naive T cell (TN) phenotype. A higher gene set score (upregulated in TEM relative to downregulated in TN) indicates an increase in the expression of the TEM phenotype, whereas a lower gene set score (upregulated in TEM relative to downregulated in TN) indicates an increase in the expression of the TN phenotype. In some embodiments, the gene set score (upregulated in TEMs versus downregulated in TNs) is determined by measuring the expression of one or more genes that are upregulated in TEM cells and/or downregulated in TN cells, such as one or more genes selected from the group consisting of MYO5A, MXD4, STK3, S1PR5, GLCCI1, CCR3, SOX13, KRTAP5-2, PEA15, PARP8, RNF166, UEVLD, LIMK1, SLC6A6, SV2A, KPNA2, OSBPL7, ST7, GGA2, PI4K2A, CD68, ZAK, RORA, TGFBI, DNAJC1, JOSD1, ZFYVE28, LRP8, OSBPL3, CMIP, NAT13, TGFB1, ANTXR2, NR4A3, RDX, ADCY9, CHN1, CD300A, SCD5, PTPN22, LGALS1, RASGEF1A, GCNT1, GLUL, ABCA2, CLDND1, PAM, CLCF1, MXRA7, CLSTN3, ACOT9, METRNL, BMPR1A, LRIG1, APOBEC3G, CACNB3, RNF19A, RAB27A, FADS3, ACTN4, TBKBP1, FAM53B, MAN1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1, TARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANXA2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21, STOM, PHACTR2, GBP5, ADRB2, PIK3AP1, DUSP10, PTGDR, EOMES, MAF, TPRG1, NBEAL2, NCAPH, SLC4A4, FOSL2, RGS2, TGFBR3, MYO1F, C17orf66, CYTH3, WEE1, PYHIN1, F2R, THBS1, CD58, AUTS2, FAM129A, TNIP3, GZMA, PRR5L, PRDM1, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, ZEB2, CST7, CCL5, GZMK and KLRB1. In some embodiments, the gene set score (upregulated in TEMs relative to downregulated in TNs) is determined using RNA sequencing, such as single cell RNA sequencing (scRNA-seq), such as illustrated in Example 7 with respect to Fig. 26C. In some embodiments, the gene set score (upregulated in TEMs relative to downregulated in TNs) is calculated using the average log-normalized gene expression value for all genes in the gene set.

For gene set score values (e.g., median gene set score values), if the positive gene set score is decreased by 100%, the result is 0. If the negative gene set score is increased by 100%, the result is 0. For example, in Fig. 25A, the median gene set score for the sample on day 1 is -0.084; the median gene set score for the sample on day 9 is 0.035; and the median gene set score for the original sample is -0.1. In Fig. 25A, increasing the median gene set score for the original sample by 100% results in a gene set score value of 0; and increasing the median gene set score for the original sample by 200% results in a gene set score value of 0.1. In Fig. 25A, decreasing the median gene set score for the sample on day 9 by 100% results in a gene set score value of 0; and a 200% reduction in the median gene set score for the day 9 sample results in a gene set score of -0.035.

As used herein, the term "bead" refers to a discrete particle with a solid surface that ranges in size from about 0.1 μm to several millimeters in diameter. The beads may be spherical (e.g., microspheres) or irregularly shaped. The beads may comprise a variety of materials, including, but not limited to, paramagnetic materials, ceramics, plastic, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, Sepharose™, cellulose, nylon, and the like. In some embodiments, the beads are relatively uniform spherical superparamagnetic polystyrene beads of approximately 4.5 μm in diameter, e.g., coated with a mixture of antibodies to CD3 (e.g., CD3 epsilon) and CD28, e.g., covalently linked thereto. In some embodiments, the beads are Dynabeads®. In some embodiments, both the anti-CD3 antibody and the anti-CD28 antibody are bound to the same bead, mimicking T cell stimulation by antigen-presenting cells. The property of Dynabeads® and the use of Dynabeads® for isolating and expanding cells are well known in the art, e.g., see, Neurauter et al., Cell isolation and expansion using Dynabeads , Adv Biochem Eng Biotechnol. 2007;106:41-73, incorporated herein by reference in its entirety.

As used herein, the term "nanomatrix" refers to a nanostructure comprising an array of flexible polymer chains. The nanomatrix has a size of 1 to 500 nm, such as 10 to 200 nm. In some embodiments, the array of flexible polymer chains is attached to one or more agonists that transmit activation signals to T cells, such as agonists that are anti-CD3 antibodies and/or anti-CD28 antibodies. In some embodiments, the nanomatrix comprises a colloidal polymer nanomatrix attached, such as covalently attached, to an agonist of one or more stimulatory molecules and/or an agonist of one or more costimulatory molecules. In some embodiments, the agonist of the one or more stimulatory molecules is a CD3 agonist (e.g., an agonist antibody to CD3). In some embodiments, the agonist of one or more costimulatory molecules is a CD28 agonist (e.g., an anti-CD28 agonist antibody). In some embodiments, the nanomatrix is characterized by the absence of a solid surface, such as a point of attachment for agonists such as anti-CD3 antibodies and/or anti-CD28 antibodies. In some embodiments, the nanomatrix is the nanomatrix disclosed in WO2014/048920A1 or provided in the MACS® GMP TransAct™ T-Cell Kit from Miltenyi Biotec GmbH, incorporated herein by reference in their entirety. The MACS® GMP TransAct™ T-Cell Kit consists of a colloidal polymeric nanomatrix covalently attached to humanized recombinant anti-human CD3 and anti-human CD28 agonist antibodies.

Various embodiments of the compositions and methods presented herein are described in more detail below. Additional definitions are set forth throughout the text of this description.

Description

Provided herein are compositions and methods for use in treating a disease, such as cancer, using cells expressing one or more chimeric antigen receptors (CARs). In some embodiments, the present invention provides a cell (e.g., an immune effector cell, such as a T cell or NK cell) engineered to express one or more CARs, wherein the CAR-T cell ("CAR-T") or CAR-NK cell exhibits an anti-tumor property.

In some embodiments, the cell expresses at least two CARs. In some embodiments, the cell expresses a first CAR that binds a first antigen and a second CAR that binds a second antigen. In some embodiments, the first antigen and the second antigen are different. In some embodiments, the first antigen is BCMA. In some embodiments, the first CAR is a CAR for BCMA that comprises a CDR, VH, VL, scFv, or CAR sequence disclosed herein, such as a sequence disclosed in Tables 3-15, 19, 20, 22, and 26, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, such as a CAR for BCMA disclosed herein. In some embodiments, the second antigen is CD19. In some embodiments, the second antigen is a CD19 CAR comprising a CDR, VH, VL, scFv, or CAR sequence disclosed herein, such as a sequence disclosed in Tables 2, 19, and 22, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, such as a CD19 CAR disclosed herein. In some embodiments, the first CAR and the second CAR are expressed by nucleic acid sequences located on a single nucleic acid molecule. In some embodiments, the nucleic acid sequence encoding the first CAR and the nucleic acid sequence encoding the second CAR are separated by a nucleic acid sequence encoding a self-cleavage site, such as a P2A site, a T2A site, an E2A site, or an F2A site. In some embodiments, the cell is a cell expressing dual CARs disclosed herein. In some embodiments, the first CAR and the second CAR are expressed by nucleic acid sequences located on separate nucleic acid molecules. In some embodiments, the cell is engineered using the cotransduction system disclosed herein.

In some embodiments, the cell expresses a CAR that binds to a first antigen and a second antigen. In some embodiments, the CAR is a diabody format CAR disclosed herein. In some embodiments, the CAR comprises a binding domain that comprises a first VH (VH1), a first VL (VL1), a second VH (VH2), and a second VL (VL2). In some embodiments, VH1 and VL1 bind to the first antigen, and VH2 and VL2 bind to the second antigen. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from N-terminus to C-terminus: VH1 - optionally Linker 1 ("L1") - VH2 - optionally Linker 2 ("L2") - VL2 - optionally Linker 3 ("L3") - VL1, VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1, VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1, VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1, VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2, VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2, VL2 - optional L1 - VH1 - optional L2 - VL1 - optional L3 - VH2 or VL2 - optional L1 - VL1 - optional L2 - VH1 - optional L3 - VH2.

In some embodiments, in a CAR of the present invention, the antigen-binding domain is combined with an intracellular signaling molecule. For example, in some embodiments, the intracellular signaling molecule comprises, but is not limited to, the CD3 zeta chain, the 4-1BB and CD28 signaling modules, and combinations thereof.

In addition, the present invention provides CAR-based compositions and their use in medicaments or methods for treating, among other diseases, cancer or any malignant neoplasm, or autoimmune diseases.

Chimeric antigen receptor (CAR)

The present invention provides immune effector cells (e.g., T cells, NK cells) engineered to contain one or more CARs that direct the immune effector cells to a cancer. This is achieved by an antigen-binding domain in the CAR that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs described herein: (1) cancer-associated antigens that are expressed on the surface of cancer cells; and (2) cancer-associated antigens that are intracellular in themselves, but fragments (peptides) of such an antigen are presented on the surface of cancer cells by the MHC (major histocompatibility complex).

Accordingly, an immune effector cell, such as one obtained by the method described herein, can be engineered to comprise a CAR that targets one of the following cancer-associated antigens (tumor antigens): CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Ag Tn, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, VEGFR2, Lewis antigen Y, CD24, PDGFR-beta, PRSS21, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostasis, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6 and E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, mutant p53, prostein, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, mutant Ras, hTERT, sarcoma breakpoint translocation antigens, ML-IAP, ERG (TMPRSS2-ETS fusion product), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, reverse transcriptase inhibitor human telomerase transcriptase, RU1, RU2, intestinal carboxylesterase and a mutant version of hsp70-2.

Sequences of non-limiting examples of various components that may be part of a CAR molecule described herein are listed in Table 1 , where "a. a." refers to amino acids and "n. a." refers to nucleic acids that encode the corresponding peptide.

Table 1. Sequences of the various CAR components SEQ ID NO Description Subsequence SEQ ID NO: 11 EF-1α gene promoter
(n.k.) CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT TTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTG CCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCC GCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC GGTTTTTGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCG TGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGTGAGTCACCCACACAAA GGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCA CACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA SEQ ID NO:1 Leadership sequence (a.k.a.) MALPVTALLLPLALLLHAARP SEQ ID NO:12 Leadership sequence (LS) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC SEQ ID NO: 199 Leadership sequence (LS) Atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggccc SEQ ID NO:351 Leadership sequence (a.k.a.) MLLLVTSLLLCELPHPAFLLIP SEQ ID NO:352 Leadership sequence (LS) atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatccca SEQ ID NO:353 Leadership sequence (LS) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCC SEQ ID NO:2 CD8 hinge region (a.k.) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO:13 CD8 hinge region (n.c.) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT SEQ ID NO:3 Hinge region Ig4 (a. k.) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM SEQ ID NO:14 Ig4 hinge region (n.c.) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCAGCAGCATCGAG AAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACT ACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG SEQ ID NO:4 IgD hinge region (a. k.) RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLLTLPRS LWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH SEQ ID NO:15 IgD hinge region (h.r.) AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGG GCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCC CTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGA GGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT SEQ ID NO:6 CD8 transmembrane domain (a.k.) IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO:17 CD8 transmembrane domain (n.c.) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC SEQ ID NO:7 Intracellular domain 4-1BB (a. k) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO:18 Intracellular domain 4-1BB (n.c.) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG SEQ ID NO:8 CD27 (a. k.) QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO:19 CD27 (n.c.) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC SEQ ID NO:9 CD3-zeta (a.k.)
(with Q/K mutation) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:20 CD3-zeta (n.c.)
(with Q/K mutation) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC SEQ ID NO: 10 CD3-zeta (a.k.)
(reference sequence NM_000734.3 in NCBI) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:21 CD3-zeta (n.c.)
(reference sequence NM_000734.3 in NCBI) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC SEQ ID NO:36 CD28 intracellular domain (amino acid sequence) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO:37 CD28 intracellular domain (nucleotide sequence) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC SEQ ID NO:38 ICOS intracellular domain (amino acid sequence) T K K K Y S S S V H D P N G E Y M F M R A V N T A K K S R L T D V T L SEQ ID NO:39 ICOS intracellular domain (nucleotide sequence) ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA SEQ ID NO:5 Hinge region/linker GS (a. k.) GGGGSGGGGS SEQ ID NO:16 Hinge region/linker GS (n.c.) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC SEQ ID NO:40 Hinge region/linker GS (n.c.) GGTGGCGGAGGTTCTGGAGGTGGGGGTTCC SEQ ID NO:25 Linker GGGGS SEQ ID NO:26 Linker (Gly-Gly-Gly-Gly-Ser)n, where n=1-6, for example, GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS SEQ ID NO:27 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:28 Linker GGGGSGGGGSGGGGS SEQ ID NO:29 Linker GGGS SEQ ID NO:41 Linker (Gly-Gly-Gly-Ser)n where n is a positive integer equal to or greater than 1 SEQ ID NO:42 Linker (Gly-Gly-Gly-Ser)n, where n=1-10, for example, GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS SEQ ID NO:43 Linker GSTSGSGKPGSGEGSTKG SEQ ID NO:30 Poly(A) (A) ₅₀₀₀
This sequence can span 50-5000 adenine bases. SEQ ID NO:31 Poly(T) (T) ₁₀₀ SEQ ID NO:32 Poly(T) (T) ₅₀₀₀
This sequence can span 50-5000 thymine bases. SEQ ID NO:33 Poly(A) (A) ₅₀₀₀
This sequence can span 100-5000 adenine bases. SEQ ID NO:34 Poly(A) (A) ₄₀₀
This sequence can span 100-400 adenine bases. SEQ ID NO:35 Poly(A) (A) ₂₀₀₀
This sequence can span 50-2000 adenine bases. SEQ ID NO:22 CAR based on PD1 (a.k.) pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvra rrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavh trgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqn qlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr SEQ ID NO:23 PD-1-based CAR (n.c.)
(ECD PD1 underlined) atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgat aatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtga ctcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcg cagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagca tgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaag cttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctata agcaggggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgca gaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc SEQ ID NO:24 PD-1-based CAR with signal sequence (a.k.a.)
(ECD PD1 underlined) Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrv tqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpea crpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapay kqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr

In some embodiments, the antigen-binding domain comprises an extracellular domain or an anti-ligand-binding fragment thereof from a molecule that binds an anti-ligand on the surface of a target cell.

The immune effector cells may comprise a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that specifically binds to a tumor antigen, e.g., a tumor antigen described herein, and an intracellular signaling domain. The intracellular signaling domain may comprise a costimulatory signaling domain and/or a primary signaling domain, e.g., a zeta chain. As described elsewhere, the methods described herein may involve transducing a cell, e.g., from a population depleted of regulatory T cells, with a nucleic acid encoding a CAR, e.g., a CAR described herein.

In some embodiments, the CAR comprises an scFv domain, wherein the scFv may be preceded by an optional leader sequence, such as that set forth in SEQ ID NO: 1, and may be followed by an optional hinge region sequence, such as that set forth in SEQ ID NO: 2, or SEQ ID NO: 36, or SEQ ID NO: 38, a transmembrane region, such as that set forth in SEQ ID NO: 6, an intracellular signaling domain that comprises SEQ ID NO: 7 or SEQ ID NO: 16, and a CD3 zeta sequence that comprises SEQ ID NO: 9 or SEQ ID NO: 10, for example, wherein the domains are contiguous and in the same reading frame to form a single fusion protein.

In some embodiments, exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain (e.g., an antigen-binding domain described herein), a hinge region (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In some embodiments, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen-binding domain (e.g., an antigen-binding domain described herein), a hinge region (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and/or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

An exemplary leader sequence is set forth as SEQ ID NO: 1. Further exemplary leader sequences include those set forth as SEQ ID NO: 351 or encoded by those set forth as SEQ ID NO: 352 or 353. An exemplary hinge/spacer sequence is set forth as SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38. An exemplary transmembrane domain sequence is set forth as SEQ ID NO: 6. An exemplary intracellular signaling domain sequence of the 4-1BB protein is set forth as SEQ ID NO: 7. An exemplary intracellular signaling domain sequence of CD27 is set forth as SEQ ID NO: 16. An exemplary CD3-zeta domain sequence is set forth as SEQ ID NO: 9 or SEQ ID NO: 10.

In some embodiments, the immune effector cell comprises a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen-binding domain, wherein the sequence is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that can be used in a CAR includes, but is not limited to, one or more intracellular signaling domains such as CD3-zeta, CD28, CD27, 4-1BB, etc. In some cases, a CAR can comprise any combination of CD3-zeta, CD28, 4-1BB, etc.

Nucleic acid sequences encoding the desired molecules can be obtained by recombinant methods known in the art, such as by screening libraries of cells expressing the nucleic acid molecule, by obtaining the nucleic acid molecule from a vector known to contain it, or by direct isolation from cells and tissues containing it using standard techniques. Alternatively, the nucleic acid of interest can be obtained by synthesis rather than cloning.

Nucleic acids encoding CARs can be introduced into immune effector cells using, for example, retroviral or lentiviral vector constructs.

Nucleic acids encoding a CAR can also be introduced into an immune effector cell using, for example, an RNA construct that can be directly introduced into the cell by transfection. A method for producing mRNA for use in transfection comprises providing for in vitro transcription (IVT) of a template using specially designed primers, followed by the addition of poly(A) to produce a construct comprising 3' and 5' untranslated region ("UTR") sequences (e.g., the 3' and/or 5' UTRs described herein), a 5' cap (e.g., the 5' cap described herein), and/or an internal ribosome entry site (IRES) (e.g., the IRES described herein), the nucleic acid to be expressed, and a poly(A) tail, typically 50-2000 bases in length (e.g., as described in the examples, e.g., under SEQ ID NO: 35). The RNA obtained in this manner can be efficiently transfected into various cell types. In some embodiments, the template comprises sequences for a CAR. In some embodiments, the RNA-based vector encoding the CAR is introduced by transduction into a cell, such as a T cell, using electroporation.

Antigen binding domain

In some embodiments, a plurality of immune effector cells, such as a population depleted of regulatory T cells, comprise a nucleic acid encoding a CAR that comprises a target-specific binding element, otherwise referred to as an antigen-binding domain. The choice of binding element depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected such that it recognizes a ligand that serves as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can serve as ligands for the antigen-binding domain in a CAR described herein include markers associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

In some embodiments, the antigen-binding domain portion of the CAR comprises an antigen-binding domain that targets a tumor antigen, such as a tumor antigen described herein.

The antigen-binding domain may be any domain that binds to an antigen, including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a variable heavy chain (VH) domain, a variable light chain (VL) domain, and a variable domain (VHH) of a camelid nanobody, as well as an alternative framework structure that is known in the art to function as an antigen-binding domain, such as a recombinant fibronectin domain, a T-cell receptor (TCR), or a fragment thereof, such as a single-chain TCR, etc. In some cases, it is desirable that the antigen-binding domain is derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be desirable for the antigen-binding domain of a CAR to comprise human or humanized residues in the antigen-binding domain of an antibody or antibody fragment.

CAR for CD19

In some embodiments, a cell expressing a CAR described herein is a cell expressing a CAR for CD19 (e.g., a cell expressing a CAR that binds to human CD19).

In some embodiments, the antigen-binding domain of a CD19 CAR has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997). In some embodiments, the antigen-binding domain of a CD19 CAR comprises the scFv fragment described in Nicholson et al. Mol. Immun . 34(16-17):1157-1165 (1997).

In some embodiments, a CD19 CAR comprises an antigen binding domain (e.g., a humanized antigen binding domain) according to Table 3 of WO2014/153270, incorporated herein by reference. WO2014/153270 also describes methods for analyzing the binding and efficacy of various CAR constructs.

In some embodiments, the parent sequence of the mouse scFv is the CAR19 construct disclosed in PCT publication WO2012/079000 (incorporated herein by reference). In some embodiments, the CD19 binding domain is the scFv described in WO2012/079000.

In some embodiments, the CAR molecule comprises a fusion polypeptide sequence as set forth in SEQ ID NO: 12 of PCT publication WO2012/079000, which provides a murine-derived scFv fragment that specifically binds to human CD19.

In some embodiments, a CAR for CD19 comprises the amino acid sequence set forth in SEQ ID NO: 12 of PCT publication WO2012/079000.

In some embodiments, the amino acid sequence is

Diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsgggg sggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdyw gqgtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeee ggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 292), or a sequence substantially homologous thereto.

In some embodiments, the CD19 CAR has the USAN designation tisagenlecleucel-T. In some embodiments, CTL019 is produced by gene modification of T cells mediated by stable insertion by transduction with a replication-defective, self-inactivating lentiviral (LV) vector comprising the CTL019 transgene under the control of the EF-1 alpha gene promoter. CTL019 may be a mixture of transgene-positive and -negative T cells that are delivered to the subject based on the percentage of transgene-positive T cells.

In other embodiments, a CAR for CD19 comprises an antigen binding domain (e.g., a humanized antigen binding domain) according to Table 3 of WO2014/153270, incorporated herein by reference.

Humanization of the anti-mouse CD19 antibody is required for clinical settings where mouse-specific moieties can induce a human anti-mouse antibody (HAMA) response in patients receiving CART19 therapy, i.e., treatment with T cells transduced with a CAR19 construct. The production, characterization, and performance of humanized CD19 CAR sequences are described in International Application WO2014/153270, incorporated herein by reference in its entirety, including Examples 1-5 (pp. 115-159).

In some embodiments, the CAR molecule is a humanized CAR for CD19 comprising the following amino acid sequence: eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 293)

In some embodiments, the CAR molecule is a humanized CAR for CD19 comprising the following amino acid sequence:

eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsgggg sggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdyw gqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeee ggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (sEQ ID NO: 294)

In some embodiments, the CAR molecule is a humanized CAR for CD19 comprising the following amino acid sequence:

MALPVTALLLPLALLLHAARP EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 349)

In some embodiments, the CAR molecule is a humanized CAR for CD19 comprising the following amino acid sequence:

EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 350)

Any known CD19 CAR can be used in accordance with the present invention, for example, a CD19 antigen-binding domain of any known CD19 CAR. For example, the CD19 CAR LG-740 is described in U.S. Patent No. 8,399,645; U.S. Patent No. 7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122 (25):4129-39 (2013) and 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10.

Illustrative CD19 CARs include the CD19 CAR described herein or the CD19 CAR described in Xu et al. Blood 123.24(2014):3750-9; Kochenderfer et al. Blood 122.25(2013):4129-39, Cruz et al. Blood 122.17(2013):2965-73, NCT00586391, NCT01087294, NCT02456350, NCT00840853, NCT02659943, NCT02650999, NCT02640209, NCT01747486, NCT02546739, NCT02656147, NCT02772198, NCT00709033, NCT02081937, NCT00924326, NCT02735083, NCT02794246, NCT02746952, NCT01593696, NCT02134262, NCT01853631, NCT02443831, NCT02277522, NCT02348216, NCT02614066, NCT02030834, NCT02624258, NCT02625480, NCT02030847, NCT02644655, NCT02349698, NCT02813837, NCT02050347, NCT01683279, NCT02529813, NCT02537977, NCT02799550, NCT02672501, NCT02819583, NCT02028455, NCT01840566, NCT01318317, NCT01864889, NCT02706405, NCT01475058, NCT01430390, NCT02146924, NCT02051257, NCT02431988, NCT01815749, NCT02153580, NCT01865617, NCT02208362, NCT02685670, NCT02535364, NCT02631044, NCT02728882, NCT02735291, NCT01860937, NCT02822326, NCT02737085, NCT02465983, NCT02132624, NCT02782351, NCT01493453, NCT02652910, NCT02247609, NCT01029366, NCT01626495, NCT02721407, NCT01044069, NCT00422383, NCT01680991, NCT02794961, or NCT02456207, each of which is incorporated herein by reference in its entirety.

In some embodiments, the CD19 CAR comprises a sequence, such as a CDR, VH, VL, scFv, or the entire CAR sequence disclosed in Table 2, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

Table 2. Amino acid sequences of exemplary CD19 binding molecules SEQ ID NO Region Subsequence CTL019 295 HCDR1 (Kabat) DYGVS 296 HCDR2 (Kabat) VIWGSETTYYNSALKS 297 HCDR3 (Kabat) HYYYGGSYAMDY 298 LCDR1 (Kabat) RASQDISKYLN 299 LCDR2 (Kabat) HTSRLHS 300 LCDR3 (Kabat) QQGNTLPYT 301 Complete amino acid sequence of CTL019 MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgg gtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyy yggsyamdywgqgtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrf peeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr 302 Complete nucleotide sequence of CTL019 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctggggagacagagtcaccatcagttgcaggcaagtcaggacattagtaaatatttaaattggtatcagcagaaacc agatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggagggg ggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccag cctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattac tacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctgga cttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttc cagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaagg aagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 303 scFv domain CTL019 Diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsgggg sevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss Humanized CAR2 295 HCDR1 (Kabat) DYGVS 304 HCDR2 (Kabat) VIWGSETTYYQSSLKS 297 HCDR3 (Kabat) HYYYGGSYAMDY 298 LCDR1 (Kabat) RASQDISKYLN 299 LCDR2 (Kabat) HTSRLHS 300 LCDR3 (Kabat) QQGNTLPYT 293 scFv-domain of CAR2 - a. k. (linker underlined) EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGG SQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS 305 scFv-domain CAR2 - n. c. GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCA GGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAAAGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGA AGCCAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGAGTGTCTCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACC AATCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC 306 CAR2 - full - a.k. MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ GTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYY YGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 307 CAR2 - full - n.c. atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcc cggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagg gcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacag ccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactat tatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttga cttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcc cagaggagggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcaga aagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 349 CAR 2A -
complete amino acid sequence; signal peptide underlined MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ GTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYY YGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 225 CAR 2A -
amino acid sequence; signal peptide absent EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 354 CAR 2A - complete nucleic acid sequence; signal peptide and stop codon are underlined atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcc cggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacaggg caccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctcttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagc caccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactatt atggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgac ttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttccca gaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaag aatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggtaa 355 CAR 2A - nucleic acid sequence; signal peptide is underlined; stop codon is absent atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcc cggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagg gcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacag ccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactat tatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttga cttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcc cagaggagggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcaga aagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 356 CAR 2A - nucleic acid sequence; signal peptide is absent; stop codon is underlined gaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatc cctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggt ccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttgggg ctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactgggga cagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgg gcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggagggaaggcg gctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcccca agaggggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggtaa SEQ ID NO:417 CAR 2A - nucleic acid sequence; signal peptide is absent; stop codon is absent gaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatc cctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtggg tccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttgg ggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattattatggcgggagctacgcaatggattactggg gacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacattt gggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggagggaggaagg cggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcc ccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 357 VH CAGGTCCAGCTGCAGGAATCAGGACCAGGGCTGGTGAAACCTAGCGAAACTCTGAGTCTGACTTGTACCGTCTCCGGGGTGTCTCTGCCAGACTACGGCGTGAGCTGGATCAGACAGCCCCCTGGCAAGTGCCTGGAGTGGATCGGCGTGATCTGGGGCTCCGAGACCACATACTATCAG AGCTCCCTGAAGTCTCGGGTGACCATCTCCCAAGGACAACTCTAAGAATCAGGTGAGCCTGAAGCTGTCTAGCGTGACCGCCGCCGATACAGCCGTGTACTATTGTGCCAAGCACTACTATTACGGCGGCTCCTATGCCATGGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT 358 VH CAGGTCCAGCTGCAGGAATCCGGCCCAGGACTGGTGAAGCCTAGCGAGACCCTGTCCCTGACCTGCACAGTGAGCGGCGTGTCCCTGCCCGATTACGGCGTGAGCTGGATCAGACAGCCCCCTGGCAAGTGTCTGGAGTGGATCGGCGTGATCTGGGGCTCTGAGACCACATACTATCAG TCCTCTCTGAAGAGCAGGGTGACCATCTCTAAGGACAACAGCAAGAATCAGGTGTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGATACAGCCGTGTACTATTGCGCCAAGCACTATTACGGCGGCTCCTATGCTATGGATTATTGGGGCAGGGCACTCTGGTCACTGTCTCATCA 359 VH CAGGTGCAGCTGCAGGAATCTGGACCCGGACTGGTGAAACCTAGTGAAACTCTGTCTCTGACTTGTACCGTCTCAGGGGTCTCACTGCCAGACTACGGCGTGTCCTGGATCAGACAGCCCCCTGGCAAGTGCCTGGAGTGGATCGGCGTGATCTGGGGCTCTGAGACCACATACTATCAG AGCTCCCTGAAGAGCCGGGTGACCATCTTCCAAGGACAACTCTAAGAATCAGGTGTCCCTGAAGCTGTCTAGCGTGACCGCCGCCGATACAGCCGTGTACTATTGTGCCAAGCACTACTATTACGGCGGCAGCTATGCCATGGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT 360 VH CAGGTCCAGCTGCAGGAAAGCGGCCCAGGACTGGTGAAGCCTAGCGAGACCCTGTCCCTGACCTGCACAGTGAGCGGCGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCCCCTGGCAAGTGTCTGGAGTGGATCGGCGTGATCTGGGGCTCCGAGACCACATACTATCAG TCCTCTCTGAAGTCTAGGGTGACAATCTCTAAGGACAACAGCAAGAATCAGGTGAGCCTGAAGCTGAGCTCCGTGACCGCAGCAGATACAGCCGTGTACTATTGTGCCAAGCACTACTATTACGGCGGCTCTTATGCTATGGATTATTGGGGGCAGGGCACTCTGGTCACTGTCTCAAGC 361 VH CAGGTGCAGCTGCAGGAGAGCGGCCCAGGACTGGTGAAGCCTTCCGAGACACTGTCTCTGACCTGTACAGTGAGCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGTGATCTGGGGCAGCGAGACCACATACTATCAG AGCTCCCTGAAGTCCAGGGTGACCATCAGCAAGGACAACTCCAAGAATCAGGTGAGCCTGAAGCTGTCTAGCGTGACCGCCGCCGATACAGCCGTGTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTATGCCATGGATTACTGGGGCCAGGGCACCCTGGTCACAGTGTCCTCT 362 VH CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCTGAGACCCTGAGCCTGACCTGCACAGTGTCCGGCGTGTCTCTGCCCGATTACGGCGTGTCCTGGATCAGACAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGTGATCTGGGGCTCTGAGACCACATACTATCAG TCTAGCCTGAAGAGCCGGGTGACAATCTCCAAGGACAACTCTAAGAATCAGGTGTCCCTGAAGCTGTCCTCTGTGACCGCCGCCGATACAGCCGTGTACTATTGTGCCAAGCACTATTACGGCGGCAGCTATGCCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCC 363 VH CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCTGAGACCCTGAGCCTGACCTGCACAGTGAGCGGCGTGTCCCTGCCCGATTACGGCGTGTCCTGGATCAGACAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGTGATCTGGGGCAGCGAGACCACATACTATCAG TCCTCTCTGAAGTCCAGGGTGACAATCTCCAAGGACAACTCTAAGAATCAGGTGAGCCTGAAGCTGAGCTCCGTGACCGCAGCAGATACAGCCGTGTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTATGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACAGTGTCTAGC 364 VH CAGGTGCAGCTGCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCCGAGACACTGTCTCTGACCTGTACAGTGTCCGGCGTGTCTCTGCCCGACTACGGCGTGAGCTGGATCAGACAGCCACCTGGCAAGGGACTGGAGTGGATCGGCGTGATCTGGGGCTCTGAGACCACATACTATCAG TCCTCTCTGAAGAGCCGGGTGACCATCAGCAAGGACAACTCCAAGAATCAGGTGTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGATACAGCCGTGTACTATTGCGCCAAGCACTATTACGGCGGCAGCTATGCCATGGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGC 365 VL GAGATCGTGATGACCCAGAGCCCAGCCACACTGAGCCTGTCCCCAGGAGAGGGCCACACTGTCTTGTAGAGCCAGCCAGGATATCTCCAAGTATCTGAACTGGTACCAGCAGAAGCCTGGACAGGCACCAAGGCTGCTGATCTACCACACCTCTAGAC TGCACAGCGGCATCCCTGCCAGGTTTTCTGGCAGCGGCTCCGGCACAGACTATACCCTGACAATCTCTAGCCTGCAGCCAGAGGATTTCGCCGTGTACTTTTGTCAGCAGGGCAATACTCTGCCATACACCTTTGGATGCGGAACTAAACTGGAAATCAAG 366 VL GAAATTGTGATGACCCAGTCCCCCGCTACTCTGTCTCTGTCCCCCGGAGAACGGGCTACTCTGTCTTGTCGCGCTTCCCAGGATATTAGCAAGTACCTGAACTGGTATCAGCAGAAGCCAGGACAGGCACCAAGGCTGCTGATCTACCACACCTCTCGCC TGCACAGCGGCATCCCTGCACGGTTCTCTGGCAGCGGCTCCGGCACAGACTACACCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTCGCCGTGTACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTTGGCTGTGGCACCAAGCTGGAGATCAAG 367 VL GAGATCGTGATGACCCAGTCCCCAGCCACACTGAGCCTGTCCCCAGGAGAGGGCCACCCTGTCTTGTAGAGCCAGCCAGGATATCTCCAAGTATCTGAACTGGTACCAGCAGAAGCCTGGACAGGCACCAAGGCTGCTGATCTACCACACCTCTAGAC TGCACAGCGGCATCCCTGCCAGGTTTTCTGGCAGCGGCTCCGGCACAGACTATACCCTGACAATCTCTAGCCTGCAGCCAGAGGATTTCGCCGTGTACTTTTGTCAGCAGGGAAATACTCTGCCATACACCTTTGGATGCGGAACTAAACTGGAAATCAAG 368 VL GAGATTGTGATGACCCAGTCCCCCGCCACCCTGAGTCTGAGCCCCGGAGAACGAGCTACCCTGAGTTGCCGAGCTTCCCAGGACATTTCCAAGTACCTGAACTGGTATCAGCAGAAGCCAGGACAGGCACCAAGGCTGCTGATCTACCACACCTCTCGCC TGCACAGCGGCATCCCAGCACGGTTCTCTGGCAGCGGCTCCGGCACAGACTACACCCTGACAATCAGCTCCCTGCAGCCTGAGGATTTCGCCGTGTACTTTTGCCAGCAGGGCAATACCCTGCCATATACATTTGGCTGTGGCACCAAGCTGGAGATCAAG 369 VL GAGATCGTGATGACCCAGTCTCCAGCCACACTGTCTCTGAGCCCAGGAGAGGGCCACCCTGTCTTGCCGCGCCAGCCAGGATATCTCCAAGTATCTGAACTGGTACCAGCAGAAGCCAGGACAGGCACCAAGGCTGCTGATCTACCACACCTCTCGCC TGCACAGCGGCATCCCAGCACGGTTCTCCGGCTCTGGCAGCGGCACAGACTACACCCTGACAATCTCCTCTCTGCAGCCCGAGGATTTCGCCGTGTATTTTTGCCAGCAGGGCAATACCCTGCCTTACACATTTGGCCAGGGCACCAAGCTGGAGATCAAG 370 VL GAGATCGTGATGACCCAGAGCCCAGCCACACTGAGCCTGTCCCCAGGAGAGAGGGCCACCCTGAGCTGCAGAGCCTCCCAGGATATCTCTAAGTATCTGAACTGGTACCAGCAGAAGCCTGGACAGGCACCAAGGCTGCTGATCTACCACACCAGCAGAC TGCACTCCGGCATCCCTGCAAGGTTCTCTGGCAGCGGCTCCGGCACAGACTACACCCTGACAATCTCTAGCCTGCAGCCTGAGGATTTCGCCGTGTATTTTTGTCAGCAGGGCAATACCCTGCCATACACATTTGGCCAGGGCACCAAGCTGGAGATCAAG 371 VL GAGATCGTGATGACCCAGAGCCCAGCCACACTGTCTCTGAGCCCAGGAGAGAGGGCCACCCTGAGCTGTCGCGCCTCCCAGGATATCTCTAAGTATCTGAACTGGTACCAGCAGAAGCCAGGACAGGCACCAAGGCTGCTGATCTACCACACCAGCCGCC TGCACTCCGGCATCCCAGCACGGTTCTCCGGCTCTGGCAGCGGCACAGACTACACCCTGACAATCTCCTCTCTGCAGCCCGAGGATTTCGCCGTGTATTTTTGCCAGCAGGGCAATACCCTGCCTTACACATTTGGCCAGGGCACCAAGCTGGAGATCAAG 372 VL GAGATCGTGATGACCCAGTCTCCAGCCACACTGAGCCTGTCCCCAGGAGAGGGCCACCCTGTCTTGCAGAGCCAGCCAGGATATCTCCAAGTATCTGAACTGGTACCAGCAGAAGCCTGGACAGGCACCAAGGCTGCTGATCTACCACACCTCTAGAC TGCACAGCGGCATCCCAGCAAGGTTCTCTGGCAGCGGCTCCGGCACAGACTACACCCTGACAATCTCTAGCCTGCAGCCTGAGGATTTCGCCGTGTATTTTTGCCAGCAGGGCAATACCCTGCCATACACATTTGGCCAGGGCACCAAGCTGGAGATCAAG SEQ ID NO: 250 VH for CD19 QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:251 VL for CD19 EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK SEQ ID NO:331 VH QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:332 VL EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIK 373 scFvCAR 1 EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGG SQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS 374 scFv CAR 3 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssg gggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 375 scFvCAR 4 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssg gggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 376 scFvCAR 5 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsg gggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 377 scFvCAR 6 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsg gggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 378 scFvCAR 7 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggg gsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 379 scFvCAR 8 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggg gsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 380 scFvCAR9 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsg gggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 381 scFvCAR 10 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggg gsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 382 scFvCAR 11 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsgggg sqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 383 scFvCAR 12 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssg gggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 384 CAR A - complete nucleotide sequence; with leader sequence atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagacatccagatgacacagactacatcctccctgtctgcctctctggggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaa ccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggg gggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctgg attcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaac attattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggac cttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgc cccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaag gaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 385 CAR A - complete amino acid transgene sequence; with leader sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK HYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 386 CAR A - scFv nucleotide sequence for CD19 with leader sequence atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagacatccagatgacacagactacatcctccctgtctgcctctctggggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaa actcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctg gatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggt agtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca 387 CAR A - amino acid sequence of scFv for CD19; with leader sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTS GSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVS 388 CAR A - complete nucleotide sequence; leader sequence is absent gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcc catcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccgg atctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagta atatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggact actggggtcaaggaacctcagtcaccgtctcctcagcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggt ggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttc gcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaacc ctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 389 CAR A - complete amino acid transgene sequence; leader sequence is absent DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 390 CAR A - scFv nucleotide sequence for CD19; leader sequence is absent gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaagg ttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctctggatccggcaagcccggatctggcgagggatcca ccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatacta taattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca 391 CAR A - amino acid sequence of scFv for CD19; leader sequence is missing DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGS TKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 392 CAR B - complete nucleotide sequence; with leader sequence ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGT ATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACA CTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGG CGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACA GCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGA GGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGG ATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCC AGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG 393 CAR B - complete transgenic amino acid sequence; with leader sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNT LPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 394 CAR B - scFv nucleotide sequence for CD19 with leader sequence ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAA GCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCG GCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGC AGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC 395 CAR B - amino acid sequence of scFv for CD19; with leader sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTS GSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 396 CAR B - complete nucleotide sequence; leader sequence is absent GACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCA CAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCAC CTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGC CCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACT GCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTG CTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTG AAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGC CTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG 397 CAR B - complete amino acid transgene sequence; leader sequence is absent DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGS TSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 398 CAR B - scFv sequence for CD19; leader sequence is missing GACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGG TTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCA CCAAGGGCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC 399 CAR B - scFv sequence for CD19; leader sequence is missing DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGS TKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

CAR for BCMA

In some embodiments, a cell expressing a CAR described herein is a cell expressing a CAR for BCMA (e.g., a cell expressing a CAR that binds to human BCMA). Exemplary CARs for BCMA can include the sequences disclosed in Table 1 or 16 of WO2016/014565, incorporated herein by reference. A CAR construct for BCMA can comprise an optional leader sequence; an optional hinge domain, e.g., a CD8 hinge domain; a transmembrane domain, e.g., a CD8 transmembrane domain; an intracellular domain, e.g., a 4-1BB intracellular domain; and a functional signaling domain, e.g., a CD3-zeta domain. In certain embodiments, the domains are contiguous and in the same reading frame, forming a single fusion protein. In other embodiments, the domains are in separate polypeptides, such as in the RCAR molecule described herein.

In some embodiments, a CAR molecule for BCMA comprises one or more CDRs, VH, VL, scFv, or full-length sequences of BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-15, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, BCMA_EBB-C1978-A4, BCMA_EBB-C1978-G1, BCMA_EBB-C1979-C1, BCMA_EBB-C1978-C7, BCMA_EBB-C1978-D10, BCMA_EBB-C1979-C12, BCMA_EBB-C1980-G4, BCMA_EBB-C1980-D2, BCMA_EBB-C1978-A10, BCMA_EBB-C1978-D4, BCMA_EBB-C1980-A2, BCMA_EBB-C1981-C3, BCMA_EBB-C1978-G4, A7D12.2, C11D5.3, C12A3.2 or C13F12.1 disclosed in WO2016/014565, or a sequence substantially (e.g. 95-99%) identical thereto.

Additional exemplary BCMA targeting sequences that can be used in BCMA CAR constructs are disclosed in WO 2017/021450, WO 2017/011804, WO 2017/025038, WO 2016/090327, WO 2016/130598, WO 2016/210293, WO 2016/090320, WO 2016/014789, WO 2016/094304, WO 2016/154055, WO 2015/166073, WO 2015/188119, WO 2015/158671, US 9243058, US 8920776, US 9273141, US 7083785, US 9034324, US 2007/0049735, US 2015/0284467, US 2015/0051266, US 2015/0344844, US 2016/0131655, US 2016/0297884, US 2016/0297885, US 2017/0051308, US 2017/0051252, US 2017/0051252, WO 2016/020332, WO 2016/087531, WO 2016/079177, WO 2015/172800, WO 2017/008169, US 9340621, US 2013/0273055, US 2016/0176973, US 2015/0368351, US 2017/0051068, US 2016/0368988, and US 2015/0232557, all incorporated by reference in their entireties. In some embodiments, additional exemplary CAR constructs for BCMA are prepared using the VH and VL sequences of PCT publication WO2012/0163805 (the contents of which are incorporated by reference herein in their entirety).

In some embodiments, CARs for BCMA comprise a sequence, e.g., a CDR, VH, VL, scFv, or the entire CAR sequence disclosed in Tables 3-15, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. In one aspect, the antigen-binding domain comprises a human antibody or a human antibody fragment. In some embodiments, the human BCMA binding domain comprises one or more (e.g., all three) of CDR1 LC, CDR2 LC, and CDR3 LC of the human BCMA binding domain described herein (e.g., Tables 3-15) and/or one or more (e.g., all three) of CDR1 HC, CDR2 HC, and CDR3 HC of the human BCMA binding domain described herein (e.g., Tables 3-15). In some embodiments, the human BCMA binding domain comprises a human VL as described herein (e.g., in Tables 3, 7, 11, 11a, and 12) and/or a human VH as described herein (e.g., in Tables 3, 7, 11, 11a, and 12). In some embodiments, the BCMA binding domain is an scFv comprising a VL and VH with an amino acid sequence from Tables 3, 7, 11, 11a, and 12. In some embodiments, the BCMA binding domain (e.g., scFv) comprises: a VL comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, e.g., conservative substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions, e.g., conservative substitutions) of the amino acid sequence presented in Tables 3, 7, 11, 11a, and 12, or a sequence with 95-99% identity to the amino acid sequence of Tables 3, 7, 11, 11a, and 12; and/or a VH comprising an amino acid sequence having at least one, two or three modifications (e.g. substitutions, e.g. conservative substitutions), but not more than 30, 20 or 10 modifications (e.g. substitutions, e.g. conservative substitutions) of the amino acid sequence presented in Tables 3, 7, 11, 11a and 12, or a sequence with 95-99% identity to the amino acid sequence from Tables 3, 7, 11, 11a and 12.

Table 3. Amino acid and nucleic acid sequences of illustrative BCMA binding molecules obtained from PALLAS SEQ ID NO Name/
description Subsequence R1B6 SEQ ID NO:44 HCDR1 (Kabat) SYAMS SEQ ID NO:45 HCDR2 (Kabat) AISGSGGSTYYADSVKG SEQ ID NO:46 HCDR3 (Kabat) REWVPYDVSWYFDY SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:48 HCDR2 (Chothia) SGSGGS SEQ ID NO:46 HCDR3 (Chothia) REWVPYDVSWYFDY SEQ ID NO:49 HCDR1 (IMGT) GFTFSSYA SEQ ID NO:50 HCDR2 (IMGT) ISGSGGST SEQ ID NO:51 HCDR3 (IMGT) ARREWVPYDVSWYFDY SEQ ID NO:52 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTLVTVSS SEQ ID NO:53 DNA VH GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCG ACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGGTGCCCTACGATGTCAGCTGGTACTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:56 LCDR3 (Kabat) QQSYSTPLT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:59 LCDR3 (Chothia) SYSTPL SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:56 LCDR3 (IMGT) QQSYSTPLT SEQ ID NO:61 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:62 DNA VL GACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGC TGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:64 scFv (VH-linker-VL) EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:65 DNA scFv GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCGACT CTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGGTGCCCTACGATGTCAGCTGGTACTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCCGGTGGT GGTGGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGA AGCTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:66 Complete amino acid sequence of CAR EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYS TPLTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:67 Complete DNA sequence of CAR GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTAC TATGCCGACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGGTGCCCTACGATGTCAGCTGGTACTTCGACTACTGGGGACAGGGCACTCTC GTGACTGTGTCCTCCGGTGGTGGTGGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAAC TGGTACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCA CCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATA TCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGA GGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAA TCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG R1F2 SEQ ID NO:44 HCDR1 (Kabat) SYAMS SEQ ID NO:45 HCDR2 (Kabat) AISGSGGSTYYADSVKG SEQ ID NO:68 HCDR3 (Kabat) REWWYDDWYLDY SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:48 HCDR2 (Chothia) SGSGGS SEQ ID NO:68 HCDR3 (Chothia) REWWYDDWYLDY SEQ ID NO:49 HCDR1 (IMGT) GFTFSSYA SEQ ID NO:50 HCDR2 (IMGT) ISGSGGST SEQ ID NO:69 HCDR3 (IMGT) ARREWWYDDWYLDY SEQ ID NO:70 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWYDDWYLDYWGQGTLVTVSS SEQ ID NO:71 DNA VH GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATG CCGACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGTACGACGATTGGTACCTGGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:56 LCDR3 (Kabat) QQSYSTPLT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:59 LCDR3 (Chothia) SYSTPL SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:56 LCDR3 (IMGT) QQSYSTPLT SEQ ID NO:61 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:62 DNA VL GACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGC TGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:72 scFv (VH-linker-VL) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWYDDWYLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:73 DNA scFv GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCGAC TCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGTACGACGATTGGTACCTGGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCCGGTGGTGGT GGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAG CTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:74 Complete amino acid sequence of CAR EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWYDDWYLDYWGQGTLV TVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PLTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:75 Complete DNA sequence of CAR GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTA CTATGCCGACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGTACGACGATTGGTACCTGGACTACTGGGGACAGGGCACTCTCGTGAC TGTGTCCTCCGGTGGTGGTGGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGT ACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCC CTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAG GAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAAT CCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG R1G5 SEQ ID NO:44 HCDR1 (Kabat) SYAMS SEQ ID NO:45 HCDR2 (Kabat) AISGSGGSTYYADSVKG SEQ ID NO:76 HCDR3 (Kabat) REWWGESWLFDY SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:48 HCDR2 (Chothia) SGSGGS SEQ ID NO:76 HCDR3 (Chothia) REWWGESWLFDY SEQ ID NO:49 HCDR1 (IMGT) GFTFSSYA SEQ ID NO:50 HCDR2 (IMGT) ISGSGGST SEQ ID NO:77 HCDR3 (IMGT) ARREWWGESWLFDY SEQ ID NO:78 VH EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSS SEQ ID NO:79 DNA VH GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATG CCGACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGGGAGAAAGCTGGCTGTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:56 LCDR3 (Kabat) QQSYSTPLT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:59 LCDR3 (Chothia) SYSTPL SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:56 LCDR3 (IMGT) QQSYSTPLT SEQ ID NO:61 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:62 DNA VL GACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGC TGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:80 scFv (VH-linker-VL) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:81 DNA scFv GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCGAC TCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGGGAGAAAGCTGGCTGTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCCGGTGGTGGT GGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAG CTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:82 Complete amino acid sequence of CAR EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLV TVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PLTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:83 Complete DNA sequence of CAR GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTA CTATGCCGACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGGGAGAAAGCTGGCTGTTCGACTACTGGGGACAGGGCACTCTCGTGAC TGTGTCCTCCGGTGGTGGTGGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGT ACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCC CTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAG GAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAAT CCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

Table 4. Kabat CDRs of illustrative BCMA binding molecules obtained from PALLAS Kabat HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 R1B6 SYAMS (SEQ ID NO: 44) AISGSGGSTYYADSVKG (SEQ ID NO: 45) REWVPYDVSWYFDY (SEQ ID NO: 46) RASQSISSYLN (SEQ ID NO: 54) AASSLQS (SEQ ID NO: 55) QQSYSTPLT (SEQ ID NO: 56) R1F2 SYAMS (SEQ ID NO: 44) AISGSGGSTYYADSVKG (SEQ ID NO: 45) REWWYDDWYLDY (SEQ ID NO: 68) RASQSISSYLN (SEQ ID NO: 54) AASSLQS (SEQ ID NO: 55) QQSYSTPLT (SEQ ID NO: 56) R1G5 SYAMS (SEQ ID NO: 44) AISGSGGSTYYADSVKG (SEQ ID NO: 45) REWWGESWLFDY (SEQ ID NO: 76) RASQSISSYLN (SEQ ID NO: 54) AASSLQS (SEQ ID NO: 55) QQSYSTPLT (SEQ ID NO: 56) Consensus sequence SYAMS (SEQ ID NO: 44) AISGSGGSTYYADSVKG (SEQ ID NO: 45) REWX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ WX ₇ X ₈ DY, wherein X ₁ is absent or V; X ₂ is absent or P; X ₃ is W or Y; X ₄ is G, Y or D; X ₅ is E, D or V; X ₆ is S or D; X ₇ is L or Y; and X ₈ is F or L (SEQ ID NO: 84) RASQSISSYLN (SEQ ID NO: 54) AASSLQS (SEQ ID NO: 55) QQSYSTPLT (SEQ ID NO: 56)

Table 5. Chothia CDRs of illustrative BCMA binding molecules obtained from PALLAS Chothia HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 R1B6 GFTFSSY (SEQ ID NO: 47) SGSGGS (SEQ ID NO: 48) REWVPYDVSWYFDY (SEQ ID NO: 46) SQSISSY (SEQ ID NO: 57) AAS (SEQ ID NO: 58) SYSTPL (SEQ ID NO: 59) R1F2 GFTFSSY (SEQ ID NO: 47) SGSGGS (SEQ ID NO: 48) REWWYDDWYLDY (SEQ ID NO: 68) SQSISSY (SEQ ID NO: 57) AAS (SEQ ID NO: 58) SYSTPL (SEQ ID NO: 59) R1G5 GFTFSSY (SEQ ID NO: 47) SGSGGS (SEQ ID NO: 48) REWWGESWLFDY (SEQ ID NO: 76) SQSISSY (SEQ ID NO: 57) AAS (SEQ ID NO: 58) SYSTPL (SEQ ID NO: 59) Consensus sequence GFTFSSY (SEQ ID NO: 47) SGSGGS (SEQ ID NO: 48) REWX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ WX ₇ X ₈ DY, wherein X ₁ is absent or V; X ₂ is absent or P; X ₃ is W or Y; X ₄ is G, Y or D; X ₅ is E, D or V; X ₆ is S or D; X ₇ is L or Y; and X ₈ is F or L (SEQ ID NO: 84) SQSISSY (SEQ ID NO: 57) AAS (SEQ ID NO: 58) SYSTPL (SEQ ID NO: 59)

Table 6. IMGT CDRs of illustrative BCMA binding molecules obtained from PALLAS IMGT HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 R1B6 GFTFSSYA (SEQ ID NO: 49) ISGSGGST (SEQ ID NO: 50) ARREWVPYDVSWYFDY (SEQ ID NO: 51) QSISSY (SEQ ID NO: 60) AAS (SEQ ID NO: 58) QQSYSTPLT (SEQ ID NO: 56) R1F2 GFTFSSYA (SEQ ID NO: 49) ISGSGGST (SEQ ID NO: 50) ARREWWYDDWYLDY (SEQ ID NO: 69) QSISSY (SEQ ID NO: 60) AAS (SEQ ID NO: 58) QQSYSTPLT (SEQ ID NO: 56) R1G5 GFTFSSYA (SEQ ID NO: 49) ISGSGGST (SEQ ID NO: 50) ARREWWGESWLFDY (SEQ ID NO: 77) QSISSY (SEQ ID NO: 60) AAS (SEQ ID NO: 58) QQSYSTPLT (SEQ ID NO: 56) Consensus sequence GFTFSSYA (SEQ ID NO: 49) ISGSGGST (SEQ ID NO: 50) ARREWX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ WX ₇ X ₈ DY, where X ₁ is absent or V; X ₂ is absent or P; X ₃ is W or Y; X ₄ is G, Y or D; X ₅ is E, D or V; X ₆ is S or D; X ₇ is L or Y; and X ₈ is F or L (SEQ ID NO: 85) QSISSY (SEQ ID NO: 60) AAS (SEQ ID NO: 58) QQSYSTPLT (SEQ ID NO: 56)

Table 7. Amino acid and nucleic acid sequences of illustrative B cell-derived molecules binding to BCMA SEQ ID NO Name/
description Subsequence PI61 SEQ ID NO:86 HCDR1 (Kabat) SYGMH SEQ ID NO:87 HCDR2 (Kabat) VISYDGSNKYYADSVKG SEQ ID NO:88 HCDR3 (Kabat) SGYALHDDYYGLDV SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:89 HCDR2 (Chothia) SYDGSN SEQ ID NO:88 HCDR3 (Chothia) SGYALHDDYYGLDV SEQ ID NO:90 HCDR1 (IMGT) GFTFSSYG SEQ ID NO:91 HCDR2 (IMGT) ISYDGSNK SEQ ID NO:92 HCDR3 (IMGT) GGSGYALHDDYYGLDV SEQ ID NO:93 VH QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO:94 DNA VH CAAGTGCAGCTGCAGGAATCCGGTGGCGGAGTCGTGCAGCCTGGAAGGAGCCTGAGACTCTCATGCGCCGCGTCAGGGTTCACCTTTTCCTCCTACGGGATGCATTGGGTCAGACAGGCCCCCGGAAAGGGACTCGAATGGGTGGCTGTGATCAGCTACGACGGCTCCAACAAGTACTACGCCG ACTCCGTGAAAGGCCGGTTCACTATCTCCCGGGACAACTCCAAGAACACGCTGTATCTGCAAATGAATTCACTGCGCGCGGAGGATACCGCTGTGTACTACTGCGGTGGCTCCGGTTACGCCCTGCACGATGACTATTACGGCCTTGACGTCTGGGGCCAGGGAACCCTCGTGACTGTGTCCAGC SEQ ID NO:95 LCDR1 (Kabat) TGTSSSDVGGYNYVS SEQ ID NO:96 LCDR2 (Kabat) DVSNRPS SEQ ID NO:97 LCDR3 (Kabat) SSYTSSSTLYV SEQ ID NO:98 LCDR1 (Chothia) TSSDVGGYNY SEQ ID NO:99 LCDR2 (Chothia) DVS SEQ ID NO: 100 LCDR3 (Chothia) YTSSSTYLE SEQ ID NO: 101 LCDR1 (IMGT) SSDVGGYNY SEQ ID NO:99 LCDR2 (IMGT) DVS SEQ ID NO:97 LCDR3 (IMGT) SSYTSSSTLYV SEQ ID NO: 102 VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO: 103 DNA VL CAGAGCGCACTGACTCAGCCGGCATCCGTGTCCGGTAGCCCCGGACAGTCGATTACCATCTCCTGTACCGGCACCTCCTCCGACGTGGGAGGGTACAACTACGTGTCGTGGTACCAGCAGCACCCAGGAAAGGCCCCTAAGTTGATGATCTACGATGTGTCAAACC GCCCGTCTGGAGTCTCCAACCGGTTCTCCGGCTCCAAGTCCGGCAACACCGCCAGCCTGACCATTAGCGGGCTGCAAGCCGAGGATGAGGCCGACTACTACTGCTCGAGCTACACATCCTCGAGCACCCTCTACGTGTTCGGCTCGGGGACTAAGGTCACCGTGCTG SEQ ID NO: 104 Linker GGGGSGGGGSGGGGS SEQ ID NO: 105 scFv (VH-linker-VL) QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSG GGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO: 106 DNA scFv CAAGTGCAGCTGCAGGAATCCGGTGGCGGAGTCGTGCAGCCTGGAAGGAGCCTGAGACTCTCATGCGCCGCGTCAGGGTTCACCTTTTCCTCCTACGGGATGCATTGGGTCAGACAGGCCCCCGGAAAGGGACTCGAATGGGTGGCTGTGATCAGCTACGACGGCTCCAACAAGTACTACGCCGAC TCCGTGAAAGGCCGGTTCACTATCTCCCGGGACAACTCCAAGAACACGCTGTATCTGCAAATGAATTCACTGCGCGCGGAGGATACCGCTGTGTACTACTGCGGTGGCTCCGGTTACGCCCTGCACGATGACTATTACGGCCTTGACGTCTGGGGCCAGGGAACCCTCGTGACTGTGTCCAGCGGTG GAGGAGGTTCGGGCGGAGGAGGATCAGGAGGGGGTGGATCGCAGAGCGCACTGACTCAGCCGGCATCCGTGTCCGGTAGCCCCGGACAGTCGATTACCATCTCCTGTACCGGCACCTCCTCCGACGTGGGAGGGTACAACTACGTGTCGTGGTACCAGCAGCACCCAGGAAAGGCCCCTAAGTTGAT GATCTACGATGTGTCAAACCGCCCGTCTGGAGTCTCCAACCGGTTCTCCGGCTCCAAGTCCGGCAACACCGCCAGCCTGACCATTAGCGGGCTGCAAGCCGAGGATGAGGCCGACTACTACTGCTCGAGCTACACATCCTCGAGCACCCTCTACGTGTTCGGCTCGGGGACTAAGGTCACCGTGCTG SEQ ID NO: 107 Complete amino acid sequence of CAR QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTL VTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSST LYVFGSGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 108 Complete DNA sequence of CAR CAAGTGCAGCTGCAGGAATCCGGTGGCGGAGTCGTGCAGCCTGGAAGGAGCCTGAGACTCTCATGCGCCGCGTCAGGGTTCACCTTTTCCTCCTACGGGATGCATTGGGTCAGACAGGCCCCCGGAAAGGGACTCGAATGGGTGGCTGTGATCAGCTACGACGGCTCCAACAAGTAC TACGCCGACTCCGTGAAAGGCCGGTTCACTATCTCCCGGGACAACTCCAAGAACACGCTGTATCTGCAAATGAATTCACTGCGCGCGGAGGATACCGCTGTGTACTACTGCGGTGGCTCCGGTTACGCCCTGCACGATGACTATTACGCCTTGACGTCTGGGGCCAGGGAACCCTC GTGACTGTGTCCAGCGGTGGAGGAGGTTCGGGCGGAGGAGGATCAGGAGGGGGTGGATCGCAGAGCGCACTGACTCAGCCGGCATCCGTGTCCGGTAGCCCCGGACAGTCGATTACCATCTCCTGTACCGGCACCTCCTCCGACGTGGGAGGGTACAACTACGTGTCGTGGTACCAG CAGCACCCAGGAAAGGCCCCTAAGTTGATGATCTACGATGTGTCAAACCGCCCGTCTGGAGTCTCCAACCGGTTCTCCGGCTCCAAGTCCGGCAACACCGCCAGCCTGACCATTAGCGGGCTGCAAGCCGAGGATGAGGCCGACTACTACTACTGCTCGAGCTACACATCCTCGAGCACC CTCTACGTGTTCGGCTCGGGGACTAAGGTCACCGTGCTGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAG GAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAAT CCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACcaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg B61-02 SEQ ID NO:86 HCDR1 (Kabat) SYGMH SEQ ID NO: 109 HCDR2 (Kabat) VISYKGSNKYYADSVKG SEQ ID NO:88 HCDR3 (Kabat) SGYALHDDYYGLDV SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO: 110 HCDR2 (Chothia) SYKGS SEQ ID NO:88 HCDR3 (Chothia) SGYALHDDYYGLDV SEQ ID NO:90 HCDR1 (IMGT) GFTFSSYG SEQ ID NO: 111 HCDR2 (IMGT) ISYKGSNK SEQ ID NO:92 HCDR3 (IMGT) GGSGYALHDDYYGLDV SEQ ID NO: 112 VH QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYKGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO: 113 DNA VH CAAGTGCAGCTTGTCGAATCGGGAGGCGGAGTGGTGCAGCCTGGACGATCGCTCCGGCTCTCATGTGCCGCGAGCGGATTCACCTTCTCGAGCTACGGCATGCACTGGGTCAGACAAGCCCCAGGAAAGGGCCTGGAATGGGTGGCTGTCATCTCGTACAAGGGCTCAAACAAGTACTACGCCG ACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGATAACTCCAAGAATACCCTCTATCTGCAAATGAACAGCCTGAGGGCCGAGGATACTGCAGTGTACTACTGCGGGGGTTCAGGCTACGCGCTGCACGACGACTACTACGGATTGGACGTCTGGGGCCAAGGAACTCTTGTGACCGTGTCCTCT SEQ ID NO:95 LCDR1 (Kabat) TGTSSSDVGGYNYVS SEQ ID NO: 114 LCDR2 (Kabat) EVSNRLR SEQ ID NO: 115 LCDR3 (Kabat) SSYTSSSALYV SEQ ID NO:98 LCDR1 (Chothia) TSSDVGGYNY SEQ ID NO: 116 LCDR2 (Chothia) EVS SEQ ID NO: 117 LCDR3 (Chothia) YTSSSALLY SEQ ID NO: 101 LCDR1 (IMGT) SSDVGGYNY SEQ ID NO: 116 LCDR2 (IMGT) EVS SEQ ID NO: 115 LCDR3 (IMGT) SSYTSSSALYV SEQ ID NO: 118 VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRLRGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSALYVFGSGTKVTVL SEQ ID NO: 119 DNA VL CAGAGCGCGCTGACTCAGCCTGCCTCCGTGAGCGGTTCGCCGGGACAGTCCATTACCATTTCGTGCACCGGGACCTCCTCCGACGTGGGAGGCTACAACTACGTGTCCTGGTACCAGCAGCATCCCGGAAAGGCCCCGAAGCTGATGATCTACGAAGTGTCGAACA GACTGCGGGGAGTCTCCAACCGCTTTTCCGGGTCCAAGTCCGGCAACACCGCCAGCCTGACCATCAGCGGGCTCCAGGCAGAAGATGAGGCTGACTATTACTGCTCCTCCTACACGTCAAGCTCCGCCCTCTACGTGTTCGGGTCCGGGACCAAAGTCACTGTGCTG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 120 scFv (VH-linker-VL) QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYKGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRLRGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSALYVFGSGTKVTVL SEQ ID NO: 121 DNA scFv CAAGTGCAGCTTGTCGAATCGGGAGGCGGAGTGGTGCAGCCTGGACGATCGCTCCGGCTCTCATGTGCCGCGAGCGGATTCACCTTCTCGAGCTACGGCATGCACTGGGTCAGACAAGCCCCAGGAAAGGGCCTGGAATGGGTGGCTGTCATCTCGTACAAGGGCTCAAACAAGTACTACGCCGACTCCG TGAAGGGCCGGTTCACCATCTCCCGCGATAACTCCAAGAATACCCTCTATCTGCAAATGAACAGCCTGAGGGCCGAGGATACTGCAGTGTACTACTGCGGGGGTTCAGGCTACGCGCTGCACGACGACTACTACGGATTGGACGTCTGGGGCCAAGGAACTCTTGTGACCGTGTCCTCTGGTGGAGGCGGA TCAGGGGGTGGCGGATCTGGGGGTGGTGGTTCCGGGGGAGGAGGATCGCAGAGCGCGCTGACTCAGCCTGCCTCCGTGAGCGGTTCGCCGGGACAGTCCATTACCATTTCGTGCACCGGGACCTCCTCCGACGTGGGAGGCTACAACTACGTGTCCTGGTACCAGCAGCATCCCGGAAAGGCCCCGAAGC TGATGATCTACGAAGTGTCGAACAGACTGCGGGGAGTCTCCAACCGCTTTTCCGGGTCCAAGTCCGGCAACACCGCCAGCCTGACCATCAGCGGGCTCCAGGCAGAAGATGAGGCTGACTATTACTGCTCCTCCTACACGTCAAGCTCCGCCCTCTACGTGTTCGGGTCCGGGACCAAAGTCACTGTGCTG SEQ ID NO: 122 Complete amino acid sequence of CAR QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYKGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLV TVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRLRGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTS SSALYVFGSGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 123 Complete DNA sequence of CAR CAAGTGCAGCTTGTCGAATCGGGAGGCGGAGTGGTGCAGCCTGGACGATCGCTCCGGCTCTCATGTGCCGCGAGCGGATTCACCTTCTCGAGCTACGGCATGCACTGGGTCAGACAAGCCCCAGGAAAGGGCCTGGAATGGGTGGCTGTCATCTCGTACAAGGGCTCAAACAAGTACT ACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGATAACTCCAAGAATACCCTCTATCTGCAAATGAACAGCCTGAGGGCCGAGGATACTGCAGTGTACTACTGCGGGGGTTCAGGCTACGCGCTGCACGACGACTACTACGGATTGGACGTCTGGGGCCAAGGAACTCTTGTG ACCGTGTCCTCTGGTGGAGGCGGATCAGGGGGTGGCGGATCTGGGGGTGGTGGTTCCGGGGGAGGAGGATCGCAGAGCGCGCTGACTCAGCCTGCCTCCGTGAGCGGTTCGCCGGGACAGTCCATTACCATTTCGTGCACCGGGACCTCCTCCGACGTGGGAGGCTACAACTACGTGTC CTGGTACCAGCAGCATCCCGGAAAGGCCCCGAAGCTGATGATCTACGAAGTGTCGAACAGACTGCGGGGAGTCTCCAACCGCTTTTCCGGGTCCAAGTCCGGCAACACCGCCAGCCTGACCATCAGCGGGCTCCAGGCAGAAGATGAGGCTGACTATTACTGCTCCTCCTACACGTCAA GCTCCGCCCTCTACGTGTTCGGGTCCGGGACCAAAGTCACTGTGCTGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGC GATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGA GGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGA ATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG B61-10 SEQ ID NO:86 HCDR1 (Kabat) SYGMH SEQ ID NO: 109 HCDR2 (Kabat) VISYKGSNKYYADSVKG SEQ ID NO:88 HCDR3 (Kabat) SGYALHDDYYGLDV SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO: 110 HCDR2 (Chothia) SYKGS SEQ ID NO:88 HCDR3 (Chothia) SGYALHDDYYGLDV SEQ ID NO:90 HCDR1 (IMGT) GFTFSSYG SEQ ID NO: 111 HCDR2 (IMGT) ISYKGSNK SEQ ID NO:92 HCDR3 (IMGT) GGSGYALHDDYYGLDV SEQ ID NO: 112 VH QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYKGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO: 113 DNA VH CAAGTGCAGCTTGTCGAATCGGGAGGCGGAGTGGTGCAGCCTGGACGATCGCTCCGGCTCTCATGTGCCGCGAGCGGATTCACCTTCTCGAGCTACGGCATGCACTGGGTCAGACAAGCCCCAGGAAAGGGCCTGGAATGGGTGGCTGTCATCTCGTACAAGGGCTCAAACAAGTACTACGCCG ACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGATAACTCCAAGAATACCCTCTATCTGCAAATGAACAGCCTGAGGGCCGAGGATACTGCAGTGTACTACTGCGGGGGTTCAGGCTACGCGCTGCACGACGACTACTACGGATTGGACGTCTGGGGCCAAGGAACTCTTGTGACCGTGTCCTCT SEQ ID NO:95 LCDR1 (Kabat) TGTSSSDVGGYNYVS SEQ ID NO: 114 LCDR2 (Kabat) EVSNRLR SEQ ID NO:97 LCDR3 (Kabat) SSYTSSSTLYV SEQ ID NO:98 LCDR1 (Chothia) TSSDVGGYNY SEQ ID NO: 116 LCDR2 (Chothia) EVS SEQ ID NO: 100 LCDR3 (Chothia) YTSSSTYLE SEQ ID NO: 101 LCDR1 (IMGT) SSDVGGYNY SEQ ID NO: 116 LCDR2 (IMGT) EVS SEQ ID NO:97 LCDR3 (IMGT) SSYTSSSTLYV SEQ ID NO: 124 VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRLRGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO: 125 DNA VL CAGAGCGCGCTGACTCAGCCTGCCTCCGTGAGCGGTTCGCCGGGACAGTCCATTACCATTTCGTGCACCGGGACCTCCTCCGACGTGGGAGGCTACAACTACGTGTCCTGGTACCAGCAGCATCCCGGAAAGGCCCCGAAGCTGATGATCTACGAAGTGTCGAACA GACTGCGGGGAGTCTCCAACCGCTTTTCCGGGTCCAAGTCCGGCAACACCGCCAGCCTGACCATCAGCGGGCTCCAGGCAGAAGATGAGGCTGACTATTACTGCTCCTCCTACACGTCAAGCTCCACCCTCTACGTGTTCGGGTCCGGGACCAAAGTCACTGTGCTG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 126 scFv (VH-linker-VL) QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYKGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRLRGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO: 127 DNA scFv CAAGTGCAGCTTGTCGAATCGGGAGGCGGAGTGGTGCAGCCTGGACGATCGCTCCGGCTCTCATGTGCCGCGAGCGGATTCACCTTCTCGAGCTACGGCATGCACTGGGTCAGACAAGCCCCAGGAAAGGGCCTGGAATGGGTGGCTGTCATCTCGTACAAGGGCTCAAACAAGTACTACGCCGACTCCG TGAAGGGCCGGTTCACCATCTCCCGCGATAACTCCAAGAATACCCTCTATCTGCAAATGAACAGCCTGAGGGCCGAGGATACTGCAGTGTACTACTGCGGGGGTTCAGGCTACGCGCTGCACGACGACTACTACGGATTGGACGTCTGGGGCCAAGGAACTCTTGTGACCGTGTCCTCTGGTGGAGGCGGA TCAGGGGGTGGCGGATCTGGGGGTGGTGGTTCCGGGGGAGGAGGATCGCAGAGCGCGCTGACTCAGCCTGCCTCCGTGAGCGGTTCGCCGGGACAGTCCATTACCATTTCGTGCACCGGGACCTCCTCCGACGTGGGAGGCTACAACTACGTGTCCTGGTACCAGCAGCATCCCGGAAAGGCCCCGAAGC TGATGATCTACGAAGTGTCGAACAGACTGCGGGGAGTCTCCAACCGCTTTTCCGGGTCCAAGTCCGGCAACACCGCCAGCCTGACCATCAGCGGGCTCCAGGCAGAAGATGAGGCTGACTATTACTGCTCCTCCTACACGTCAAGCTCCACCCTCTACGTGTTCGGGTCCGGGACCAAAGTCACTGTGCTG SEQ ID NO: 128 Complete amino acid sequence of CAR QVQLVESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYKGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLV TVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRLRGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTS SSTLYVFGSGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 129 Complete DNA sequence of CAR CAAGTGCAGCTTGTCGAATCGGGAGGCGGAGTGGTGCAGCCTGGACGATCGCTCCGGCTCTCATGTGCCGCGAGCGGATTCACCTTCTCGAGCTACGGCATGCACTGGGTCAGACAAGCCCCAGGAAAGGGCCTGGAATGGGTGGCTGTCATCTCGTACAAGGGCTCAAACAAGTACT ACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGATAACTCCAAGAATACCCTCTATCTGCAAATGAACAGCCTGAGGGCCGAGGATACTGCAGTGTACTACTGCGGGGGTTCAGGCTACGCGCTGCACGACGACTACTACGGATTGGACGTCTGGGGCCAAGGAACTCTTGTG ACCGTGTCCTCTGGTGGAGGCGGATCAGGGGGTGGCGGATCTGGGGGTGGTGGTTCCGGGGGAGGAGGATCGCAGAGCGCGCTGACTCAGCCTGCCTCCGTGAGCGGTTCGCCGGGACAGTCCATTACCATTTCGTGCACCGGGACCTCCTCCGACGTGGGAGGCTACAACTACGTGTC CTGGTACCAGCAGCATCCCGGAAAGGCCCCGAAGCTGATGATCTACGAAGTGTCGAACAGACTGCGGGGAGTCTCCAACCGCTTTTCCGGGTCCAAGTCCGGCAACACCGCCAGCCTGACCATCAGCGGGCTCCAGGCAGAAGATGAGGCTGACTATTACTGCTCCTCCTACACGTCAA GCTCCACCCTCTACGTGTTCGGGTCCGGGACCAAAGTCACTGTGCTGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGC GATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGA GGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGA ATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

Table 8. Kabat CDRs of illustrative B-cell-derived BCMA-binding molecules Kabat HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 PI61 SYGMH (SEQ ID NO: 86) VISYDGSNKYYADSVKG (SEQ ID NO: 87) SGYALHDDYYGLDV (SEQ ID NO: 88) TGTSSDVGGYNYVS (SEQ ID NO: 95) DVSNRPS (SEQ ID NO: 96) SSYTSSSTLYV (SEQ ID NO: 97) B61-02 SYGMH (SEQ ID NO: 86) VISYKGSNKYYADSVKG (SEQ ID NO: 109) SGYALHDDYYGLDV (SEQ ID NO: 88) TGTSSDVGGYNYVS (SEQ ID NO: 95) EVSNRLR (SEQ ID NO: 114) SSYTSSSALYV (SEQ ID NO: 115) B61-10 SYGMH (SEQ ID NO: 86) VISYKGSNKYYADSVKG (SEQ ID NO: 109) SGYALHDDYYGLDV (SEQ ID NO: 88) TGTSSDVGGYNYVS (SEQ ID NO: 95) EVSNRLR (SEQ ID NO: 114) SSYTSSSTLYV (SEQ ID NO: 97) Consensus sequence SYGMH (SEQ ID NO: 86) VISYXGSNKYYADSVKG, where X is D or K (SEQ ID NO: 130) SGYALHDDYYGLDV (SEQ ID NO: 88) TGTSSDVGGYNYVS (SEQ ID NO: 95) X ₁ VSNRX ₂ X ₃ , where X ₁ is D or E; X ₂ is P or L; and X ₃ is S or R (SEQ ID NO: 131) SSYTSSSXLYV, where X is T or A (SEQ ID NO: 132)

Table 9. Chothia CDRs of illustrative B-cell-derived BCMA-binding molecules Chothia HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 PI61 GFTFSSY (SEQ ID NO: 47) SYDGSN (SEQ ID NO: 89) SGYALHDDYYGLDV (SEQ ID NO: 88) TSSDVGGYNY (SEQ ID NO: 98) DVS (SEQ ID NO: 99) YTSSSTLY (SEQ ID NO: 100) B61-02 GFTFSSY (SEQ ID NO: 47) SYKGSN (SEQ ID NO: 110) SGYALHDDYYGLDV (SEQ ID NO: 88) TSSDVGGYNY (SEQ ID NO: 98) EVS (SEQ ID NO: 116) YTSSSALY (SEQ ID NO: 117) B61-10 GFTFSSY (SEQ ID NO: 47) SYKGSN (SEQ ID NO: 110) SGYALHDDYYGLDV (SEQ ID NO: 88) TSSDVGGYNY (SEQ ID NO: 98) EVS (SEQ ID NO: 116) YTSSSTLY (SEQ ID NO: 100) Consensus sequence GFTFSSY (SEQ ID NO: 47) SYXGSN, where X is D or K (SEQ ID NO: 133) SGYALHDDYYGLDV (SEQ ID NO: 88) TSSDVGGYNY (SEQ ID NO: 98) XVS, where X is D or E (SEQ ID NO: 134) YTSSSXLY, where X is T or A (SEQ ID NO: 135)

Table 10. IMGT CDRs of illustrative B-cell-derived BCMA-binding molecules IMGT HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 PI61 GFTFSSYG (SEQ ID NO: 90) ISYDGSNK (SEQ ID NO: 91) GGSGYALHDDYYGLDV (SEQ ID NO: 92) SSDVGGYNY (SEQ ID NO: 101) DVS (SEQ ID NO: 99) SSYTSSSTLYV (SEQ ID NO: 97) B61-02 GFTFSSYG (SEQ ID NO: 90) ISYKGSNK (SEQ ID NO: 111) GGSGYALHDDYYGLDV (SEQ ID NO: 92) SSDVGGYNY (SEQ ID NO: 101) EVS (SEQ ID NO: 116) SSYTSSSALYV (SEQ ID NO: 115) B61-10 GFTFSSYG (SEQ ID NO: 90) ISYKGSNK (SEQ ID NO: 111) GGSGYALHDDYYGLDV (SEQ ID NO: 92) SSDVGGYNY (SEQ ID NO: 101) EVS (SEQ ID NO: 116) SSYTSSSTLYV (SEQ ID NO: 97) Consensus sequence GFTFSSYG (SEQ ID NO: 90) ISYXGSNK, where X is D or K (SEQ ID NO: 136) GGSGYALHDDYYGLDV (SEQ ID NO: 92) SSDVGGYNY (SEQ ID NO: 101) XVS, where X is D or E (SEQ ID NO: 134) SSYTSSSXLYV, where X is T or A (SEQ ID NO: 132)

Table 11. Amino acid and nucleic acid sequences of exemplary BCMA binding molecules based on PI61 Identification Protein sequence DNA sequence (5'-3') Signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) Atggccctccctgtcaccgctctgttgctgccgcttgctctgctgctccacgcagcgcgaccg (SEQ ID NO: 252) VHPI61 QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS (SEQ ID NO: 93) CAGGTACAATTGCAGGAGTCTGGAGGCGGTGTGGTGCAACCCGGTCGCAGCTTGCGCCTGAGTTGTGCTGCGTCTGGATTTACATTTTCATCTTACGGAATGCATTGGGTACGCCAGGCACCGGGGAAAGGCCTTGAATGGGTGGCTGTAATTTCATACGATGGTTCCAACAAATACTATGCTG ACTCAGTCAAGGGTCGATTTACAATTAGTCGGGACAACTCCAAGAACACCCTTTATCTTCAAATGAATTCCCTTAGAGCAGAGGATACGGCGGTCTATTACTGTGGTGGCAGTGGTTATGCACTTCATGATGATTACTATGGCTTGGATGTCTGGGGGCAAGGGACGCTTGTAACTGTATCCTCT (SEQ ID NO: 260) VLPI61 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL (SEQ ID NO: 102) caatctgctctgactcaaccagcaagcgtatcagggtcaccggacagagtattaccataagttgcacggggacctctagcgatgtaggggggtataattatgtatcttggtatcaacaacaccccgggaaagcccctaaattgatgatctacgacgtgagcaatc gacctagtggcgtatcaaatcgcttctctggtagcaagagtgggaatacggcgtcccttactattagcggattgcaagcagaagatgaggccgattactactgcagctcctatactagctcttctacattgtacgtctttgggagcggaacaaaagtaacagtactc (SEQ ID NO: 261) Linker GGGGSGGGGSGGGGS (SEQ ID NO: 104) ScFvPI61 QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSG GGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL (SEQ ID NO: 105) CaggtacaattgcaggagtctggaggcggtgtgGtgcaacccggtcgcagcttgcgcctgagttgtGctgcgtctggatttacattttcatct tacggaAtgcattgggtacgccaggcaccggggaaaggcCttgaatgggtggctgtaatttcatacgatggtTccaacaaatactatgctgac tcagtcaagggtCgatttacaattagtcgggacaactccaagaacAccctttatcttcaaatgaattcccttagagcaGaggatacggcggtctattactgtggtggcagtGgttatgcacttcatgatgattactatggcttgGatgtctgggggcaagggacgcttgtaactgtaTcctctggtg gtggtggtagtggtgggggaggcTccggcggtggcggctctcaatctgctctgactCaaccagcaagcgtatcagggtcaccgggacagAgtattaccataagttgcacggggacctctagcGatgtaggggggtataattatgtatcttggtatCaacaacaccccgggaaagcccctaaattgat gAtctacgacgtgagcaatcgacctagtggcgtaTcaaatcgcttctctggtagcaagagtgggaatAcggcgtcccttactattagcggattgcaagcaGaagatgaggccgattactactgcagctcctatActagctcttctacattgtacgtctttgggagcggaacaaaagtaacagtactc (SEQ ID NO: 253) Transmembrane domain and hinge region TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 202) AcaacaacacctgccccgagaccgcctacaccaGccccgactattgccagccagcctctgagcctcAggcctgaggcctgtaggcccgcagcggcggcGcag ttcatacacggggcttggatttcgcttgtGatatttatatttgggctcctttggcggggacaTgtggcgtgctgcttctgtcacttgttattacactgtactgt (SEQ ID NO: 254) 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7) AaacgcgggcgaaaaaaattgctgtatatttttAagcagccatttatgaggcccgttcagacgacgCaggaggacggttgctcttgcaggttcccagaagaggaagaagggggctgtgaattg (SEQ ID NO: 255) CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) CgggttaaattttcaagatccgcagacgctccaGcataccaacagggacaaaaccaactctataacGagctgaatcttggaagaagggaggaatatgatGtgctggataaacggcgcggtagagatccggagAtgggcggaaaaccaaggcgaaaaaaccctcagGag ggactctacaacgaactgcagaaagacaaaAtggcggaggcttattccgaaataggcatgaagGgcgagcggaggcgagggaaagggcacgacggaCtgtatcaaggcctctcaaccgcgactaaggatAcgtacgacgccctgcacatgcaggccctgcctccgaga (SEQ ID NO: 256) Complete design of CAR PI61 MALPVTALLLPLALLLHAARPQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYA LHDDYYGLDVWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAD YYCSSYTSSSTLYVFGSGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 257) ATGGCCCTCCCTGTCACCGCTCTGTTGCTGCCGCTTGCTCTGCTGCTCCACGCAGCGCGACCGCAGGTACAATTGCAGGAGTCTGGAGGCGGTGTGGTGCAACCCGGTCGCAGCTTGCGCCTGAGTTGTGCTGCGTCTGGATTTACATTTTCATCTTACGGAATGCATTGGGTACGCCAGGCAC CGGGGAAAGGCCTTGAATGGGTGGCTGTAATTTCATACGATGGTTCCAACAAATACTATGCTGACTCAGTCAAGGGTCGATTTACAATTAGTCGGGACAACTCCAAGAACACCCTTTATCTTCAAATGAATTCCCTTAGAGCAGAGGATACGGCGGTCTATTACTGTGGTGGCAGTGGTTATGCA CTTCATGATGATTACTATGGCTTGGATGTCTGGGGGCAAGGGACGCTTGTAACTGTATCCTCTGGTGGTGGTGGTAGTGGTGGGGGAGGCTCCGGCGGTGGCGGCTCTCAATCTGCTCTGACTCAACCAGCAAGCGTATCAGGGTCACCGGGACAGAGTATTACCATAAGTTGCACGGGGACCTC TAGCGATGTAGGGGGGTATAATTATGTATCTTGGTATCAACAACACCCCGGGAAAGCCCCTAAATTGATGATCTACGACGTGAGCAATCGACCTAGTGGCGTATCAAATCGCTTCTCTGGTAGCAAGAGTGGGAATACGGCGTCCCTTACTATTAGCGGATTGCAAGCAGAAGATGAGGCCGATT ACTACTGCAGCTCCTATACTAGCTCTTCTACATTGTACGTCTTTGGGAGCGGAACAAAAGTAACAGTACTCACAACAACACCTGCCCCGAGACCGCCTACACCAGCCCCGACTATTGCCAGCCAGCCTCTGAGCCTCAGGCCTGAGGCCTGTAGGCCCGCAGCGGGCGGCGCAGTTCATACACGG GGCTTGGATTTCGCTTGTGATATTTATATTTGGGCTCCTTTGGCGGGGACATGTGGCGTGCTGCTTCTGTCACTTGTTATTACACTGTACTGTAAACGCGGGCGAAAAAAATTGCTGTATATTTTTAAGCAGCCATTTATGAGGCCCGTTCAGACGACGCAGGAGGAGGACGGTTGCTCTTGCAG GTTCCCAGAAGAGGAAGAAGGGGGCTGTGAATTGCGGGTTAAATTTTCAAGATCCGCAGACGCTCCAGCATACCAACAGGGACAAAACCAACTCTATAACGAGCTGAATCTTGGAAGAAGGGAGGAATTGATGTGCTGGATAAACGGCGCGGTAGAGATCCGGAGATGGGCGGAAAACCAAGGC GAAAAAACCCTCAGGAGGGACTCTACAACGAACTGCAGAAAGACAAAATGGCGGAGGCTTATTCCGAAATAGGCATGAAGGGCGAGCGGAGGCGAGGGAAAGGGCACGACGACTGTATCAAGGCCTCTCAACCGCGACTAAGGATACGTACGACGCCCTGCACATGCAGGCCCTGCCTCCGAGA (SEQ ID NO: 258) Complete CAR PI61 construct (nucleic acid with signal peptide and stop codons) ATGGCCCTCCCTGTCACCGCTCTGTTGCTGCCGCTTGCTCTGCTGCTCCACGCAGCGCGACCGCAGGTACAATTGCAGGAGTCTGGAGGCGGTGTGGTGCAACCCGGTCGCAGCTTGCGCCTGAGTTGTGCTGCGTCTGGATTTACATTTTCATCTTACGGAATGCATTGGGTACGCCAGGCACC GGGGAAAGGCCTTGAATGGGTGGCTGTAATTTCATACGATGGTTCCAACAAATACTATGCTGACTCAGTCAAGGGTCGATTTACAATTAGTCGGGACAACTCCAAGAACACCCTTTATCTTCAAATGAATTCCCTTAGAGCAGAGGATACGGCGGTCTATTACTGTGGTGGCAGTGGTTATGCACT TCATGATGATTACTATGGCTTGGATGTCTGGGGGCAAGGGACGCTTGTAACTGTATCCTCTGGTGGTGGTGGTAGTGGTGGGGGAGGCTCCGGCGGTGGCGGCTCTCAATCTGCTCTGACTCAACCAGCAAGCGTATCAGGGTCACCGGGACAGAGTATTACCATAAGTTGCACGGGGACCTCTA GCGATGTAGGGGGGTATAATTATGTATCTTGGTATCAACAACACCCCGGGAAAGCCCCTAAATTGATGATCTACGACGTGAGCAATCGACCTAGTGGCGTATCAAATCGCTTCTCTGGTAGCAAGAGTGGGAATACGGCGTCCCTTACTATTAGCGGATTGCAAGCAGAAGATGAGGCCGATTACT ACTGCAGCTCCTATACTAGCTCTTCTACATTGTACGTCTTTGGGAGCGGAACAAAAGTAACAGTACTCACAACAACACCTGCCCCGAGACCGCCTACACCAGCCCCGACTATTGCCAGCCAGCCTCTGAGCCTCAGGCCTGAGGCCTGTAGGCCCGCAGCGGGCGGCGCAGTTCATACACGGGGGC TTGGATTTCGCTTGTGATATTTATATTTGGGCTCCTTTGGCGGGGACATGTGGCGTGCTGCTTCTGTCACTTGTTATTACACTGTACTGTAAACGCGGGCGAAAAAAATTGCTGTATATTTTTAAGCAGCCATTTATGAGGCCCGTTCAGACGACGCAGGAGGAGGACGGTTGCTCTTGCAGGTTC CCAGAAGAGGAAGAAGGGGGCTGTGAATTGCGGGTTAAATTTTCAAGATCCGCAGACGCTCCAGCATACCAACAGGGACAAAACCAACTCTATAACGAGCTGAATCTTGGAAGAAGGGAGGAATTGATGTGCTGGATAAACGGCGCGGTAGAGATCCGGAGATGGGCGGAAAACCAAGGCGAAAA AACCCTCAGGAGGACTCTACAACGAACTGCAGAAAGACAAAATGGCGGAGGCTTATTCCGAAATAGGCATGAAGGGCGAGCGGAGGCGAGGGAAAGGGCACGACGACTGTATCAAGGCCTCTCAACCGCGACTAAGGATACGTACGACGCCCTGCACATGCAGGCCCTGCCTCCGAGATGATAA (SEQ ID NO: 416) Mature CAR PI61 protein QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTL VTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSST LYVFGSGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 107) caggtacaattgcaggagtctggaggcggtgtggtgcaacccggtcgcagcttgcgcctgagttgtgctgcgtctggatttacattttcatcttacggaatgcattgggtacgccaggcaccggggaaaggccttgaatgggtggctgtaatttcatacgatggttccaacaaatac tatgctgactcagtcaagggtcgatttacaattagtcgggacaactccaagaacaccctttatcttcaaatgaattcccttagagcagaggatacggcggtctattactgtggtggcagtggttatgcacttcatgatgattactatggcttggatgtctgggggcaagggacgctt gtaactgtatcctctggtggtggtggtagtggtgggggaggctccggcggtggcggctctcaatctgctctgactcaaccagcaagcgtatcagggtcaccgggacagtattaccataagttgcacggggacctctagcgatgtaggggggtataattatgtatcttggtatcaa caacaccccgggaaagcccctaaattgatgatctacgacgtgagcaatcgacctagtggcgtatcaaatcgcttctctggtagcaagagtgggaatacggcgtcccttactattagcggattgcaagcagaagatgaggccgattactactgcagctcctatactagctcttctaca ttgtacgtctttgggagcggaacaaaagtaacagtactcacaacaacacctgccccgagaccgcctacaccagccccgactattgccagccagcctctgagcctcaggcctgaggcctgtaggcccgcagcgggcggcgcagttcatacacggggcttggatttcgcttgtgatatt tatatttgggctcctttggcggggacatgtggcgtgctgcttctgtcacttgttattacactgtactgtaaacgcgggcgaaaaaaattgctgtatatttttaagcagccatttatgaggcccgttcagacgacgcaggaggaggacggttgctcttgcaggttcccagaagaggaa gaagggggctgtgaattgcgggttaaattttcaagatccgcagacgctccagcataccaacagggacaaaaccaactctataacgagctgaatcttggaagaagggaggaatatgatgtgctggataaacggcgcggtagagatccggagatgggcggaaaaccaaggcgaaaaaac cctcaggagggactctacaacgaactgcagaaagacaaaatggcggaggcttattccgaaataggcatgaagggcgagcggaggcgagggaaagggcacgacggactgtatcaaggcctctcaaccgcgactaaggatacgtacgacgccctgcacatgcaggccctgcctccgaga (SEQ ID NO: 259)

Table 11A. Additional illustrative sequences of PI161-based BCMA binders SEQ ID NO Region Subsequence 400 VH CAGGTGCAGCTGCAGGAGTCCGGCGGCGGCGTGGTGCAGCCAGGCCGGTCCCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTTTCCTCTTATGGCATGCACTGGGTGAGACAGGCACCTGGCAAGGGACTGGAGTGGGTGGCCGTGATCTCCTACGACGGCTCTAACAAGTATTACGCCG ATAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACTCCAAGAATACACTGTATCTGCAGATGAATAGCCTGCGGGCCGAGGATACCGCCGTGTATTACTGCGGAGGCTCCGGCTACGCACTGCACGACGATTATTACGGACTGGACGTGTGGGGACAGGGCACCCTGGTCACAGTGAGCTCC 401 VH CAGGTGCAGCTGCAGGAGTCTGGCGGAGGAGTGGTGCAGCCAGGCCGGTCCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACATTTTCTAGCTACGGAATGCACTGGGTGCGCCAGGCACCTGGCAAGGGACTGGAGTGGGTGGCCGTGATCTCCTATGACGGCTCTAACAAGTACTATGCCG ATTCCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACTCCAAGAATACACTGTACCTGCAGATGAATTCCCTGCGGGCCGAGGATACCGCCGTGTACTATTGTGGCGGCTCTGGCTATGCCCTGCACGACGATTACTATGGACTGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGTCCTCT 402 VH CAGGTGCAGCTGCAGGAGTCTGGCGGAGGAGTGGTGCAGCCAGGCCGGAGCCTGAGACTGTCCTGCGCCGCCTCTGGCTTCACCTTTAGCTCCTATGGCATGCACTGGGTGAGACAGGCACCTGGCAAGGGACTGGAGTGGGTGGCCGTGATCAGCTACGACGGCTCCAACAAGTATTACGCCG ATAGCGTGAAGGGCAGGTTCACCATCTCTCGCAAACAGCAAGAATACACTGTATCTGCAGATGAATTCCCTGCGGGCCGAGGATACAGCCGTGTATTACTGCGGAGGCAGCGGCTACGCACTGCACGACGATTATTACGGACTGGACGTGTGGGGACAGGGCACCCTGGTCACAGTGTCTAGC 403 VH CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGGTCTCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACCTTTAGCTCCTACGGAATGCACTGGGTGCGCCAGGCACCTGGCAAGGGACTGGAGTGGGTGGCCGTGATCTCTTATGACGGCAGCAACAAGTACTATGCCG ATAGCGTGAAGGGCAGGTTCACCATCTCCCCGCGACAACTCTAAGAATACACTGTACCTGCAGATGAATAGCCTGCGGGCCGAGGATACCGCCGTGTACTATTGCGGAGGCTCCGGCTATGCACTGCACGACGATTACTATGGACTGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGTCTAGC 404 VH CAGGTCCAGCTGCAGGAGAGTGGGGGGGGGGTCGTCCAGCCCGGAAGAAGCCTGAGACTGTCATGTGCCGCATCTGGGTTTTACTTTTAGCTCTATGGAATGCACTGGGTGCGCCAGGCACCTGGCAAGTGCCTGGAGTGGGTGGCCGTGATCTCCTACGACGGCTCTAACAAGTACTATGCCG ATAGCGTGAAGGGCCGGTTCACCATCAGCAGAGACAACTCCAAGAATACACTGTATCTGCAGATGAATTCTCTGCGGGCCGAGGATACCGCCGTGTACTATTGTGGAGGCTCCGGCTACGCACTGCACGACGATTACTATGGACTGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGTCTAGC 405 VH CAGGTCCAGCTGCAGGAATCCGGCGGAGGAGTGGTGCAGCCAGGCCGGTCTCTGAGACTGAGCTGCGCCGCCTCCGGCTTCACATTTTCCTCTTATGGCATGCACTGGGTGAGACAGGCCCCTGGCAAGTGTCTGGAGTGGGTGGCCGTGATCTCTTACGACGGCAGCAACAAGTATTACGCCG ATAGCGTGAAGGGCAGGTTCACCATCTCCCCGCGACAACTCTAAGAATACACTGTATCTGCAGATGAATTCCCTGCGGGCCGAGGATACCGCCGTGTATTACTGCGGCGGCTCTGGCTACGCCCTGCACGACGACTACTATGGACTGGATGTCTGGGGGCAGGGCACACTGGTCACTGTCTCTTCA 406 VH CAGGTCCAGCTGCAGGAATCAGGGGGGGGGGTCGTCCAGCCCGGAAGAAGTCTGAGACTGTCATGTGCCGCATCAGGGTTTACCTTTAGCTCCTATGGAATGCACTGGGTGCGCCAGGCACCTGGCAAGTGCCTGGAGTGGGTGGCCGTGATCTCCTACGACGGCTCTAACAAGTACTATGCCG ATAGCGTGAAGGGCCGGTTCACCATCAGCAGAGACAACTCCAAGAATACACTGTATCTGCAGATGAATTCTCTGCGGGCCGAGGATACCGCCGTGTACTATTGTGGAGGCTCCGGCTACGCACTGCACGACGATTACTATGGACTGGACGTGTGGGGACAGGGCACCCTGGTGACAGTGTCTAGC 407 VH CAGGTCCAGCTGCAGGAATCCGGCGGAGGAGTGGTGCAGCCAGGCCGGTCTCTGAGACTGAGCTGCGCCGCCTCCGGCTTCACCTTTTCCTCTTATGGCATGCACTGGGTGAGACAGGCCCCTGGCAAGTGTCTGGAGTGGGTGGCCGTGATCTCTTACGACGGCAGCAACAAGTATTACGCCG ATAGCGTGAAGGGCAGGTTCACCATCTCCCCGCGACAACTCTAAGAATACACTGTATCTGCAGATGAATTCCCTGCGGGCCGAGGATACAGCCGTGTATTACTGTGGCGGCTCTGGCTACGCCCTGCATGATGATTATTATGGACTGGATGTCTGGGGGCAGGGCACACTGGTCACTGTCTCTTCC 408 VL CAGTCTGCCCTGACCCAGCCAGCAAGCGTGTCCGGCTCTCCTGGCCAGAGCATCACAATCTCCTGCACCGGCACAAGCTCCGACGTGGGAGGCTATAACTACGTGAGCTGGTATCAGCAGCACCCAGGCAAGGCCCCCAAGCTGATGATCTACGACGTGAGCAACA GGCCTTCTGGCGTGAGCAATCGCTTCAGCGGCTCCAAGTCTGGCAATACCGCCTCTCTGACAATCAGCGGCCTGCAGGCAGAGGACGAGGCAGATTATTACTGCTCTAGCTATACCTCCTCTAGCACACTGTACGTGTTTGGCAGCGGCACCAAGGTGACAGTGCTG 409 VL CAGAGCGCCCTGACCCAGCCAGCATCCGTGTCTGGCAGCCCAGGCCAGTCTATCACAATCAGCTGCACCGGCACAAGCTCCGACGTGGGAGGCTACAACTATGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTATGACGTGAGCAACC GGCCATCCGGCGTGTCTAATAGATTCTCCGGCTCTAAGAGCGGCAATACCGCCTCCCTGACAATCTCTGGCCTGCAGGCAGAGGACGAGGCAGATTACTATTGTTCTAGCTACACCTCCTCTAGCACACTGTACGTGTTCGGCAGCGGCACCAAGGTGACAGTGCTG 410 VL CAGTCTGCCCTGACCCAGCCAGCAAGCGTGTCCGGCTCTCCTGGCCAGTCCATCACAATCTCTTGTACCGGCACATCCCTCTGACGTGGGCGGCTATAACTACGTGTCCTGGTATCAGCAGCACCCAGGCAAGGCCCCCAAGCTGATGATCTACGATGTGAGCAACA GGCCTTCTGGCGTGAGCAATCGCTTCAGCGGCTCCAAGTCTGGCAATACCGCCAGCCTGACAATCTCCGGCCTGCAGGCAGAGGACGAGGCAGATTATTACTGCAGCTCCTATACCTCTAGCTCCACACTGTACGTGTTTGGCAGCGGCACCAAGGTGACAGTGCTG 411 VL CAGAGCGCCCTGACCCAGCCAGCATCCGTGTCTGGCAGCCCAGGCCAGTCCATCACAATCTCTTGCACCGGCACATCTAGCGACGTGGGCGGCTACAACTACGTGAGCTGGTACCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTATGATGTGAGCAACC GGCCCTCCGGCGTGTCTAATAGATTCTCCGGCTCTAAGAGCGGCAATACCGCCAGCCTGACAATCTCCGGCCTGCAGGCAGAGGACGAGGCAGATTACTATTGCTCCTCTTACACCAGCTCCTCTACACTGTACGTGTTCGGCTCCGGCACCAAGGTGACAGTGCTG 412 VL CAGTCTGCCCTGACCCAGCCTGCAAGCGTGTCCGGCTCTCCAGGCCAGTCTATCACAATCAGCTGTACCGGCACAAGCTCCGACGTGGGCGGCTATAACTACGTGAGCTGGTATCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGAGCAACC GGCCCTCTGGCGTGAGCAATCGGTTCAGCGGCAGCAAGTCTGGCAATACCGCCTCCCTGACAATCTCTGGCCTGCAGGCAGAGGACGAGGCAGATTATTACTGTAGCAGTTATACTTCAAGCTCAACCCTGTACGTGTTTGGATGCGGCACTAAGGTCACCGTCCTG 413 VL CAGTCTGCTCTGACCCAGCCCGCTTCCGTCTCAGGGTCTCCAGGACAGTCAATTACCATTAGTTGCACAGGCACCTCATCCGATGTGGGCGGCTATAACTACGTGTCCTGGTATCAGCAGCACCCAGGCAAGGCCCCCAAGCTGATGATCTACGACGTGAGCAACA GGCCATCTGGCGTGAGCAATCGCTTCAGCGGCTCCAAGTCTGGCAATACCGCCAGCCTGACAATCTCCGGCCTGCAGGCAGAGGACGAGGCAGATTACTATTGCAGCTCCTATACCTCTAGCTCCACACTGTACGTGTTTGGCTGTGGCACCAAGGTGACAGTGCTG 414 VL CAGTCTGCCCTGACCCAGCCTGCAAGCGTGTCCGGCTCTCCAGGCCAGTCTATCACAATCAGCTGTACCGGCACAAGCTCCGACGTGGGCGGCTATAACTACGTGAGCTGGTATCAGCAGCACCCTGGCAAGGCCCCAAAGCTGATGATCTACGACGTGAGCAACC GGCCCTCTGGCGTGAGCAATCGGTTCAGCGGCAGCAAGTCTGGCAATACCGCCTCCCTGACAATCTCTGGCCTGCAGGCAGAGGACGAGGCAGATTATTACTGTAGCTCCTACACTTCTTCAAGCACACTGTATGTCTTTGGATGCGGAACTAAGGTCACTGTCCTG 415 VL CAGTCTGCTCTGACCCAGCCCGCTTCCGTCTCAGGATCTCCAGGACAGTCTATTACAATTAGTTGCACAGGAACCTCTTCCGATGTGGGCGGCTATAACTACGTGTCCTGGTATCAGCAGCACCCAGGCAAGGCCCCCAAGCTGATGATCTACGACGTGAGCAACA GGCCTTCTGGCGTGAGCAATCGCTTCAGCGGCTCCAAGTCTGGCAATACCGCCAGCCTGACAATCTCCGGCCTGCAGGCAGAGGACGAGGCAGATTACTATTGCAGCTCCTATACCTCTAGCTCCACACTGTACGTGTTTGGCTGTGGCACCAAGGTGACAGTGCTG SEQ ID NO:93 VH for BCMA (PI61) QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO: 102 VL for BCMA (PI61) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO:333 VH variant for BCMA (PI61) QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO:334 Option VL for BCMA (PI61) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVL

Table 12. Amino acid and nucleic acid sequences of illustrative BCMA-binding molecules derived from hybridoma SEQ ID NO Title/Description Subsequence Hy03 SEQ ID NO: 137 HCDR1 (Kabat) GFWMS SEQ ID NO: 138 HCDR2 (Kabat) NIKQDGSEKYYVDSVRG SEQ ID NO: 139 HCDR3 (Kabat) ALDYYGMDV SEQ ID NO: 140 HCDR1 (Chothia) GFTFSGF SEQ ID NO: 141 HCDR2 (Chothia) KQDGSE SEQ ID NO: 139 HCDR3 (Chothia) ALDYYGMDV SEQ ID NO: 142 HCDR1 (IMGT) GFTFSGFW SEQ ID NO: 143 HCDR2 (IMGT) IKQDGSEK SEQ ID NO: 144 HCDR3 (IMGT) ARALDYYGMDV SEQ ID NO: 145 VH EVQLVESGGGLVQPGGSLRLSCAASGTFFSGFWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARALDYYGMDVWGQGTTVTVSS SEQ ID NO: 146 DNA VH GAAGTGCAACTGGTGGAGAGCGGTGGAGGGCTTGTCCAGCCCGGAGGATCGCTGCGGCTGTCCTGTGCTGCGTCCGGGTTCACCTTCTCCGGCTTCTGGATGTCCTGGGTCAGACAGGCACCGGGAAAGGGCCTCGAATGGGTGGCCAACATCAAGCAGGATGGCTCCGAGAAGTAC TACGTCGACTCCGTGAGAGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCGCTGTACCTCCAATGAATAGCCTCAGGGCGGAAGATACTGCTGTGTATTACTGCGCACGCGCCCCTTGACTACGGCATGGACGTCTGGGGCCAAGGGACCACTGTGACCGTGTCTAGC SEQ ID NO: 147 LCDR1 (Kabat) RSSQSLLDSDDGNTYLD SEQ ID NO: 148 LCDR2 (Kabat) TLSYRAS SEQ ID NO: 149 LCDR3 (Kabat) TQRLEFPSIT SEQ ID NO: 150 LCDR1 (Chothia) SQSLLDSDDGNTY SEQ ID NO: 151 LCDR2 (Chothia) TLS SEQ ID NO: 152 LCDR3 (Chothia) RLEFPSI SEQ ID NO: 153 LCDR1 (IMGT) QSLLDSDDGNTY SEQ ID NO: 151 LCDR2 (IMGT) TLS SEQ ID NO: 149 LCDR3 (IMGT) TQRLEFPSIT SEQ ID NO: 154 VL DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDGNTYLDWYLQKPGQSPRLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCTQRLEFPSITFGQGTRLEIK SEQ ID NO: 155 DNA VL GATATCGTGATGACCCAGACTCCCCTGTCCCTGCCTGTGACTCCCGGAGAACCAGCCTCCATTTCCTGCCGGTCCTCCCAGTCCCTGCTGGACAGCGACGACGGCAACACTTACCTGGACTGGTACTTGCAGAAGCCGGGCCAATCGCCTCGCCTGCTGATCTATACCCTG TCATACCGGGCCTCAGGAGTGCCTGACCGCTTCTCGGGATCAGGGAGCGGGACCGATTTCACCCTGAAAATTTCCCGAGTGGAAGCCGAGGACGTCGGACTGTACTACTGCACCCAGCGCCTCGAATTCCCGTCGATTACGTTTGGACAGGGTACCCGGCTTGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 156 scFv (VH-linker-VL) EVQLVESGGGLVQPGGSLRLSCAASGTFFSGFWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARALDYYGMDVWGQGTTVTVSSGGGGSGGG GSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPRLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCTQRLEFPSITFGQGTRLEIK SEQ ID NO: 157 DNA scFv GAAGTGCAACTGGTGGAGAGCGGTGGAGGGCTTGTCCAGCCCGGAGGATCGCTGCGGCTGTCCTGTGCTGCGTCCGGGTTCACCTTCTCCGGCTTCTGGATGTCCTGGGTCAGACAGGCACCGGGAAAGGGCCTCGAATGGGTGGCCAACATCAAGCAGGATGGCTCCGAGAAGTACTACGTCGACTCC GTGAGAGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCGCTGTACCTCCAATGAATAGCCTCAGGGCGGAAGATACTGCTGTGTATTACTGCGCACGCGCCCTTGACTACGGCATGGACGTCTGGGGCCAAGGGACCACTGTGACCGTGTCTAGCGGAGGCGGAGGTTCAGGGGGCGGT GGATCAGGCGGAGGAGGATCGGGGGGTGGTGGATCGGATATCGTGATGACCCAGACTCCCCTGTCCCTGCCTGTGACTCCCGGAGAACCAGCCTCCATTTCCTGCCGGTCCTCCCAGTCCCTGCTGGACAGCGACGACGGCAACACTTACCTGGACTGGTACTTGCAGAAGCCGGGCCAATCGCCTCGC CTGCTGATCTATACCCTGTCATACCGGGCCTCAGGAGTGCCTGACCGCTTCTCGGGATCAGGGAGCGGGACCGATTTCACCCTGAAAATTTCCCGAGTGGAAGCCGAGGACGTCGGACTGTACTACTGCACCCAGCGCCTCGAATTCCCGTCGATTACGTTTGGACAGGGTACCCGGCTTGAGATCAAG SEQ ID NO: 158 Complete amino acid sequence of CAR EVQLVESGGGLVQPGGSLRLSCAASGTFFSGFWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARALDYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDGNTYLDWYLQKPGQSPRLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCTQRLE FPSITFGQGTRLEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 159 Complete DNA sequence of CAR GAAGTGCAACTGGTGGAGAGCGGTGGAGGGCTTGTCCAGCCCGGAGGATCGCTGCGGCTGTCCTGTGCTGCGTCCGGGTTCACCTTCTCCGGCTTCTGGATGTCCTGGGTCAGACAGGCACCGGGAAAGGGCCTCGAATGGGTGGCCAACATCAAGCAGGATGGCTCCGAGAAGTACT ACGTCGACTCCGTGAGAGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCGCTGTACCTCCAAATGAATAGCCTCAGGGCGGAAGATACTGCTGTGTATTACTGCGCACGCGCCCTTGACTACGGCATGGACGTCTGGGGCCAAGGGACCACTGTGACCGTGTCTAGCGG AGGCGGAGGTTCAGGGGGCGGTGGATCAGGCGGAGGAGGATCGGGGGGTGGTGGATCGGATATCGTGATGACCCAGACTCCCCTGTCCCTGCCTGTGACTCCCGGAGAACCAGCCTCCATTTCCTGCCGGTCCTCCCAGTCCCTGCTGGACAGCGACGACGGCAACACTTACCTGGAC TGGTACTTGCAGAAGCCGGGCCAATCGCCTCGCCTGCTGATCTATACCCTGTCATACCGGGCCTCAGGAGTGCCTGACCGCTTCTCGGATCAGGGAGCGGGACCGATTTCACCCTGAAAATTTCCCGAGTGGAAGCCGAGGACGTCGGACTGTACTACTGCACCCAGCGCCTCGAAT TCCCGTCGATTACGTTTGGACAGGGTACCCGGCTTGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGA TATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAG GAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGA ATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG Hy52 SEQ ID NO: 160 HCDR1 (Kabat) SFRMN SEQ ID NO: 161 HCDR2 (Kabat) SISSSSSSYIYYADSVKG SEQ ID NO: 162 HCDR3 (Kabat) WLSYYGMDV SEQ ID NO: 163 HCDR1 (Chothia) GFTFSSF SEQ ID NO: 164 HCDR2 (Chothia) SSSSSY SEQ ID NO: 162 HCDR3 (Chothia) WLSYYGMDV SEQ ID NO: 165 HCDR1 (IMGT) GFTFSSFR SEQ ID NO: 166 HCDR2 (IMGT) ISSSSSYI SEQ ID NO: 167 HCDR3 (IMGT) ARWLSYYGMDV SEQ ID NO: 168 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSFRMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWLSYYGMDVWGQGTTVTVSS SEQ ID NO: 169 DNA VH GAAGTGCAACTGGTGGAGAGCGGTGGAGGGCTTGTCAAGCCCGGAGGATCGCTGCGGCTGTCCTGTGCTGCGTCCGGGTTCACCTTCTCCTCGTTCCGCATGAACTGGGTCAGACAGGCACCGGGAAAGGGCCTCGAATGGGTGTCCTCAATCTCATCGTCCTCGTCCTACATCTAC TACGCCGACTCCGTGAAAGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCGCTGTACCTCCAAATGAATAGCCTCAGGGCGGAAGATACTGCTGTGTATTACTGCGCACGCTGGCTTTCCTACTACGGCATGGACGTCTGGGGCCAAGGGACCACTGTGACCGTGTCTAGC SEQ ID NO: 147 LCDR1 (Kabat) RSSQSLLDSDDGNTYLD SEQ ID NO: 170 LCDR2 (Kabat) TLSFRAS SEQ ID NO: 171 LCDR3 (Kabat) MQRIGFPIT SEQ ID NO: 150 LCDR1 (Chothia) SQSLLDSDDGNTY SEQ ID NO: 151 LCDR2 (Chothia) TLS SEQ ID NO: 172 LCDR3 (Chothia) RIGFPI SEQ ID NO: 153 LCDR1 (IMGT) QSLLDSDDGNTY SEQ ID NO: 151 LCDR2 (IMGT) TLS SEQ ID NO: 171 LCDR3 (IMGT) MQRIGFPIT SEQ ID NO: 173 VL DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDGNTYLDWYLQKPGQSPQLLIYTLSFRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQRIGFPITFGQGTRLEIK SEQ ID NO: 174 DNA VL GATATCGTGATGACCCAGACTCCCCTGTCCCTGCCTGTGACTCCCGGAGAACCAGCCTCCATTTCCTGCCGGTCCTCCCAGTCCCTGCTGGACAGCGACGACGGCAACACTTACCTGGACTGGTACTTGCAGAAGCCGGGCCAATCGCCTCAGCTGCTGATCTATACCC TGTCATTCCGGGCCTCAGGAGTGCCTGACCGCTTCTCGGGATCAGGGAGCGGGACCGATTTCACCCTGAAAATTAGGCGAGTGGAAGCCGAGGACGTCGGAGTGTACTACTGCATGCAGCGCATCGGCTTCCCGATTACGTTTGGACAGGGTACCCGGCTTGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 175 scFv (VH-linker-VL) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSFRMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWLSYYGMDVWGQGTTVTVSSGGGGSGG GGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYTLSFRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQRIGFPITFGQGTRLEIK SEQ ID NO: 176 DNA scFv GAAGTGCAACTGGTGGAGAGCGGTGGAGGGCTTGTCAAGCCCGGAGGATCGCTGCGGCTGTCCTGTGCTGCGTCCGGGTTCACCTTCTCCTCGTTCCGCATGAACTGGGTCAGACAGGCACCGGGAAAGGGCCTCGAATGGGTGTCCTCAATCTCATCGTCCTCGTCCTACATCTACTACGCCGACTC CGTGAAAGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCGCTGTACCTCCAAATGAATAGCCTCAGGGCGGAAGATACTGCTGTGTATTACTGCGCACGCTGGCTTTCCTACTACGGCATGGACGTCTGGGGCCAAGGGACCACTGTGACCGTGTCTAGCGGAGGCGGAGGTTCAGGGGGCG GTGGATCAGGCGGAGGAGGATCGGGGGGTGGTGGATCGGATATCGTGATGACCCAGACTCCCCTGTCCCTGCCTGTGACTCCCGGAGAACCAGCCTCCATTTCCTGCCGGTCCTCCCAGTCCCTGCTGGACAGCGACGACGGCAACACTTACCTGGACTGGTACTTGCAGAAGCCGGGCCAATCGCCT CAGCTGCTGATCTATACCCTGTCATTCCGGGCCTCAGGAGTGCCTGACCGCTTCTCGGGATCAGGGAGCGGGACCGATTTCACCCTGAAAATTAGGCGAGTGGAAGCCGAGGACGTCGGAGTGTACTACTGCATGCAGCGCATCGGCTTCCCGATTACGTTTGGACAGGGTACCCGGCTTGAGATCAAG SEQ ID NO: 177 Complete amino acid sequence of CAR EVQLVESGGGLVKPGGSLRLSCAASGFTFSSFRMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWLSYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDGNTYLDWYLQKPGQSPQLLIYTLSFRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQRIG FPITFGQGTRLEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 178 Complete DNA sequence of CAR GAAGTGCAACTGGTGGAGAGCGGTGGAGGGCTTGTCAAGCCCGGAGGATCGCTGCGGCTGTCCTGTGCTGCGTCCGGGTTCACCTTCTCCTCGTTCCGCATGAACTGGGTCAGACAGGCACCGGGAAAGGGCCTCGAATGGGTGTCCTCAATCTCATCGTCCTCGTCCTACATCTAC TACGCCGACTCCGTGAAAGGCCGCTTCACCATCTCCCGGGACAACGCCAAGAACTCGCTGTACCTCCAAATGAATAGCCTCAGGGCGGAAGATACTGCTGTGTATTACTGCGCACGCTGGCTTTCCTACTACGGCATGGACGTCTGGGGCCAAGGGACCACTGTGACCGTGTCTAGCG GAGGCGGAGGTTCAGGGGGCGGTGGATCAGGCGGAGGAGGATCGGGGGGTGGTGGATCGGATATCGTGATGACCCAGACTCCCCTGTCCCTGCCTGTGACTCCCGGAGAACCAGCCTCCATTTCCTGCCGGTCCTCCCAGTCCCTGCTGGACAGCGACGACGGCAACACTTACCTGGA CTGGTACTTGCAGAAGCCGGGCCAATCGCCTCAGCTGCTGATCTATACCCTGTCATTCCGGGCCTCAGGAGTGCCTGACCGCTTCTCGGGATCAGGGAGCGGGACCGATTTCACCCTGAAAATTAGGCGAGTGGAAGCCGAGGACGTCGGAGTGTACTACTGCATGCAGCGCATCGGC TTCCCGATTACGTTTGGACAGGGTACCCGGCTTGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGAT ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGG AGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACCAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAA TCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

Table 13. Kabat CDRs of illustrative hybridoma-derived BCMA-binding molecules Kabat HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Hy03 GFWMS (SEQ ID NO: 137) NIKQDGSEKYYVDSVRG (SEQ ID NO: 138) ALDYYGMDV (SEQ ID NO: 139) RSSQSLLDSDGNTYLD (SEQ ID NO: 147) TLSYRAS (SEQ ID NO: 148) TQRLEFPSIT (SEQ ID NO: 149) Hy52 SFRMN (SEQ ID NO: 160) SISSSSSYIYYADSVKG (SEQ ID NO: 161) WLSYYGMDV (SEQ ID NO: 162) RSSQSLLDSDGNTYLD (SEQ ID NO: 147) TLSFRAS (SEQ ID NO: 170) MQRIGFPIT (SEQ ID NO: 171) Consensus sequence X ₁ FX ₂ MX ₃ , wherein X ₁ is G or S; X ₂ is W or R; and X ₃ is S or N (SEQ ID NO: 179) X ₁ IX ₂ X ₃ X ₄ X ₅ SX ₆ X ₇ YYX ₈ DSVX ₉ G, wherein X ₁ is N or S; X ₂ is K or S; X ₃ is Q or S; X ₄ is D or S; X ₅ is G or S; X ₆ is E or Y; X ₇ is K or I; X ₈ is V or A; and X ₉ is R or K (SEQ ID NO: 180) X ₁ LX ₂ YYGMDV, where X ₁ is A or W; and X ₂ is D or S (SEQ ID NO: 181) RSSQSLLDSDGNTYLD (SEQ ID NO: 147) TLSXRAS, where X is Y or F (SEQ ID NO: 182) X ₁ QRX ₂ X ₃ FPX ₄ IT, where X ₁ is T or M; X ₂ is L or I; X ₃ is E or G; and X ₄ is S or absent (SEQ ID NO: 183)

Table 14. Chothia CDRs of illustrative BCMA-binding molecules derived from hybridoma Chothia HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Hy03 GFTFSGF (SEQ ID NO: 140) KQDGSE (SEQ ID NO: 141) ALDYYGMDV (SEQ ID NO: 139) SQSLLDSDGNTY (SEQ ID NO: 150) TLS (SEQ ID NO: 151) RLEFPSI (SEQ ID NO: 152) Hy52 GFTFSSF (SEQ ID NO: 163) SSSSSY (SEQ ID NO: 164) WLSYYGMDV (SEQ ID NO: 162) SQSLLDSDGNTY (SEQ ID NO: 150) TLS (SEQ ID NO: 151) RIGFPI (SEQ ID NO: 172) Consensus sequence GFTFSXF, where X is G or S (SEQ ID NO: 184) X ₁ X ₂ X ₃ X ₄ SX ₅ , wherein X ₁ is K or S; X ₂ is Q or S; X ₃ is D or S; X ₄ is G or S; and X ₅ is E or Y (SEQ ID NO: 185) X ₁ LX ₂ YYGMDV, where X ₁ is A or W; and X ₂ is D or S (SEQ ID NO: 181) SQSLLDSDGNTY (SEQ ID NO: 150) TLS (SEQ ID NO: 151) RX ₁ X ₂ FPX ₃ I, where X ₁ is L or I; X ₂ is E or G; and X ₃ is S or absent (SEQ ID NO: 186)

Table 15. IMGT CDRs of illustrative hybridoma-derived BCMA-binding molecules IMGT HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Hy03 GFTFSGFW (SEQ ID NO: 142) IKQDGSEK (SEQ ID NO: 143) ARALDYYGMDV (SEQ ID NO: 144) QSLLDSDDGNTY (SEQ ID NO: 153) TLS (SEQ ID NO: 151) TQRLEFPSIT (SEQ ID NO: 149) Hy52 GFTFSSFR (SEQ ID NO: 165) ISSSSSYI (SEQ ID NO: 166) ARWLSYYGMDV (SEQ ID NO: 167) QSLLDSDDGNTY (SEQ ID NO: 153) TLS (SEQ ID NO: 151) MQRIGFPIT (SEQ ID NO: 171) Consensus sequence GFTFSX ₁ FX ₂ , where X ₁ is G or S; and X ₂ is W or R (SEQ ID NO: 187) IX ₁ X ₂ X ₃ X ₄ SX ₅ X ₆ , wherein X ₁ is K or S; X ₂ is Q or S; X ₃ is D or S; X ₄ is G or S; X ₅ is E or Y; and X ₆ is K or I (SEQ ID NO: 188) ARX ₁ LX ₂ YYGMDV, where X ₁ is A or W; and X ₂ is D or S (SEQ ID NO: 189) QSLLDSDDGNTY (SEQ ID NO: 153) TLS (SEQ ID NO: 151) X ₁ QRX ₂ X ₃ FPX ₄ IT, where X ₁ is T or M; X ₂ is L or I; X ₃ is E or G; and X ₄ is S or absent (SEQ ID NO: 183)

Table 20. Amino acid and nucleic acid sequences of illustrative BCMA binding molecules SEQ ID NO Name/
description Subsequence duBCMA.4 SEQ ID NO:231 HCDR1 (Kabat) NHGMS SEQ ID NO:232 HCDR2 (Kabat) GIVYSGSTYYAASVKG SEQ ID NO:233 HCDR3 (Kabat) HGGESDV SEQ ID NO:234 HCDR1 (Chothia) GFALSNH SEQ ID NO:235 HCDR2 (Chothia) VYSGS SEQ ID NO:233 HCDR3 (Chothia) HGGESDV SEQ ID NO:236 HCDR1 (IMGT) GFALSNHG SEQ ID NO:237 HCDR2 (IMGT) IVYSGST SEQ ID NO:238 HCDR3 (IMGT) SAHGGESDV SEQ ID NO:239 VH EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS SEQ ID NO:262 DNA VH GAAGTGCAATTGGTGGAATCAGGGGGAGGACTTGTGCAGCCTGGAGGATCGCTGAGACTGTCATGTGCCGTGTCCGGCTTTGCCCTGTCCAACCACGGGATGTCCTGGGTCCGCCGCGCGCCTGGAAAGGGCCTCGAATGGGTGTCGGGTATTGTGTACAGCGGTAGCACCT ACTATGCCGCATCCGTGAAGGGGAGATTCACCATCAGCCGGGACAACTCCAGGAACACTCTGTACCTCCAATGAATTCGCTGAGGCCAGAGGACACTGCCATCTACTACTGCTCCGCGCATGGCGGAGAGTCCGACGTCTGGGGACAGGGGACCACGTGACCGTGTCTAGC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:240 LCDR3 (Kabat) QQSYSTPYT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:241 LCDR3 (Chothia) SYSTPY SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:240 LCDR3 (IMGT) QQSYSTPYT SEQ ID NO:242 VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK SEQ ID NO:263 DNA VL GACATCCAGCTCACCCAGTCCCCGAGCTCGCTGTCCGCCTCCGTGGGAGATCGGGTCACCATCACGTGCCGCGCCAGCCAGTCGATTTCCTCCTACCTGAACTGGTACCAACAGAAGCCCGGAAAAGCCCCGAAGCTTCTCATCTACGCCGCCTCGAGCC TGCAGTCAGGAGTGCCCTCACGGTTCTCCGGCTCCGGTTCCGGTACTGATTTCACCCTGACCATTTCCTCCCTGCAACCGGAGGACTTCGCTACTTACTACTGCCAGCAGTCGTACTCCACCCCCTACACTTTCGGACAAGGCACCAAGGTCGAAATCAAG SEQ ID NO:243 Linker ASGGGGSGGGGSGGGGS SEQ ID NO:200 scFv (VH-linker-VL) EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGG GGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK SEQ ID NO:201 DNA scFv Gaagtgcaattggtggaatcagggggaggacttgtgcagcctggaggatcgctgagactgtcatgtgccgtgtccggctttgccctgtccaaccacgggatgtcctgggtccgccgcgcgcctggaaagggcctcgaatgggtgtcgggtattgtgtacagcggtagcacctactatgc cgcatccgtgaaggggagattcaccatcagccgggacaactccaggaacactctgtacctccaaatgaattcgctgaggccagaggacactgccatctactactgctccgcgcatggcggagagtccgacgtctggggacaggggaccaccgtgaccgtgtctagcgcgtccggcggag gcggcagcgggggtggtggttcagggggcggcggatcggacatccagctcacccagtccccgagctcgctgtccgcctccgtggggagatcgggtcaccatcacgtgccgcgccagccagtcgatttcctcctacctgaactggtaccaacagaagcccggaaaagccccgaagcttctc atctacgccgcctcgagcctgcagtcaggagtgccctcacggttctccggctccggttccggtactgatttcaccctgaccatttcctccctgcaaccggaggacttcgctacttactactgccagcagtcgtactccaccccctacactttcggacaaggcaccaaggtcgaaatcaag SEQ ID NO:230 Complete amino acid sequence of CAR EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS ASGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFG QGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Table 26. CDRs of illustrative BCMA-binding molecules duBCMA.4 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Kabat SEQ ID NO:231 SEQ ID NO:232 SEQ ID NO:233 SEQ ID NO:54 SEQ ID NO:55 SEQ ID NO:240 Chothia SEQ ID NO:234 SEQ ID NO:235 SEQ ID NO:233 SEQ ID NO:57 SEQ ID NO:58 SEQ ID NO:241 IMGT SEQ ID NO:236 SEQ ID NO:237 SEQ ID NO:238 SEQ ID NO:60 SEQ ID NO:58 SEQ ID NO:240

In some embodiments, the human BCMA binding domain comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC.

In certain embodiments, a CAR molecule described herein or a BCMA binding domain described herein comprises:

(1) one, two, or three light chain (LC) CDRs selected from

(i) CDR1 LC of SEQ ID NO: 54, CDR2 LC of SEQ ID NO: 55 and CDR3 LC of SEQ ID NO: 56; and/or

(2) one, two, or three heavy chain (HC) CDRs from any of the following:

(i) CDR1 HC of SEQ ID NO: 44, CDR2 HC of SEQ ID NO: 45 and CDR3 HC of SEQ ID NO: 84; (ii) CDR1 HC of SEQ ID NO: 44, CDR2 HC of SEQ ID NO: 45 and CDR3 HC of SEQ ID NO: 46; (iii) CDR1 HC under SEQ ID NO: 44, CDR2 HC under SEQ ID NO: 45 and CDR3 HC under SEQ ID NO: 68 or (iv) CDR1 HC under SEQ ID NO: 44, CDR2 HC under SEQ ID NO: 45 and CDR3 HC under SEQ ID NO: 76.

In certain embodiments, a CAR molecule described herein or a BCMA binding domain described herein comprises:

(1) one, two, or three light chain (LC) CDRs from any of the following:

(i) CDR1 LC under SEQ ID NO: 95, CDR2 LC under SEQ ID NO: 131 and CDR3 LC under SEQ ID NO: 132; (ii) CDR1 LC of SEQ ID NO: 95, CDR2 LC of SEQ ID NO: 96 and CDR3 LC of SEQ ID NO: 97; (iii) CDR1 LC under SEQ ID NO: 95, CDR2 LC under SEQ ID NO: 114 and CDR3 LC under SEQ ID NO: 115 or (iv) CDR1 LC under SEQ ID NO: 95, CDR2 LC under SEQ ID NO: 114 and CDR3 LC under SEQ ID NO: 97; and/or

(2) one, two, or three heavy chain (HC) CDRs from any of the following:

(i) CDR1 HC of SEQ ID NO: 86, CDR2 HC of SEQ ID NO: 130 and CDR3 HC of SEQ ID NO: 88; (ii) CDR1 HC under SEQ ID NO: 86, CDR2 HC under SEQ ID NO: 87 and CDR3 HC under SEQ ID NO: 88 or (iii) CDR1 HC under SEQ ID NO: 86, CDR2 HC under SEQ ID NO: 109 and CDR3 HC under SEQ ID NO: 88.

In certain embodiments, a CAR molecule described herein or a BCMA binding domain described herein comprises:

(1) one, two, or three light chain (LC) CDRs from any of the following:

(i) CDR1 LC of SEQ ID NO: 147, CDR2 LC of SEQ ID NO: 182 and CDR3 LC of SEQ ID NO: 183; (ii) CDR1 LC under SEQ ID NO: 147, CDR2 LC under SEQ ID NO: 148 and CDR3 LC under SEQ ID NO: 149 or (iii) CDR1 LC under SEQ ID NO: 147, CDR2 LC under SEQ ID NO: 170 and CDR3 LC under SEQ ID NO: 171; and/or

(2) one, two, or three heavy chain (HC) CDRs from any of the following:

(i) CDR1 HC of SEQ ID NO: 179, CDR2 HC of SEQ ID NO: 180 and CDR3 HC of SEQ ID NO: 181; (ii) CDR1 HC under SEQ ID NO: 137, CDR2 HC under SEQ ID NO: 138 and CDR3 HC under SEQ ID NO: 139 or (iii) CDR1 HC under SEQ ID NO: 160, CDR2 HC under SEQ ID NO: 161 and CDR3 HC under SEQ ID NO: 162.

In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 84, 54, 55, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 68, 54, 55, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively.

In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 47, 48, 84, 57, 58, and 59, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 47, 48, 46, 57, 58, and 59, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 47, 48, 68, 57, 58, and 59, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 47, 48, 76, 57, 58, and 59, respectively.

In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 49, 50, 85, 60, 58, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 49, 50, 51, 60, 58, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 49, 50, 69, 60, 58, and 56, respectively. In some embodiments, CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprise the amino acid sequences of SEQ ID NOs: 49, 50, 77, 60, 58, and 56, respectively.

In some embodiments, the human BCMA binding domain comprises an scFv comprising a VH (e.g., a VH described herein) and a VL (e.g., a VL described herein). In some embodiments, the VH is attached to the VL via a linker, e.g., a linker described herein, e.g., a linker described in Table 1. In some embodiments, the human BCMA binding domain comprises a (Gly ₄ -Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be presented, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In some embodiments, the BCMA binding domain is a fragment, such as a single chain variable fragment (scFv). In some embodiments, the BCMA binding domain is an Fv, a Fab (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In some embodiments, the antibodies and fragments thereof of the present invention bind to the BCMA protein with wild-type or enhanced affinity.

In some cases, scFv can be prepared according to a method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be prepared by linking the VH and VL regions to each other using flexible polypeptide linkers. scFv molecules contain a linker (e.g., a Ser-Gly linker) with optimized length and/or amino acid composition. The length of the linker can greatly influence the folding and interaction of the variable regions of scFv. Indeed, if a short polypeptide linker (e.g. 5-10 amino acids long) is used, intrachain folding is prevented. Interchain folding is also necessary to bring the two variable regions together to form a functional epitope-binding site. For examples of linker orientation and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715, incorporated herein by reference.

The scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In some embodiments, the linker sequence comprises groups of glycine and serine repeats, such as (Gly ₄ Ser)n, wherein n is a positive integer that is equal to or greater than 1 (SEQ ID NO: 25). In some embodiments, the linker may be (Gly ₄ Ser) ₄ (SEQ ID NO: 27) or (Gly ₄ Ser) ₃ (SEQ ID NO: 28). By changing the length of the linker, activity may be maintained or enhanced, resulting in improved performance in activity assays.

CAR for CD20

In some embodiments, a cell expressing a CAR described herein is a cell expressing a CD20 CAR (e.g., a cell expressing a CAR that binds to human CD20). In some embodiments, a cell expressing a CD20 CAR comprises an antigen-binding domain of WO2016164731 and WO2018067992, incorporated herein by reference. Exemplary CD20 binding sequences or CD20 CAR sequences are disclosed, e.g., in Tables 1-5 of WO2018067992. In some embodiments, a CD20 CAR comprises a CDR, variable region, scFv, or full-length CD20 CAR sequence disclosed in WO2018067992 or WO2016164731.

CAR for CD22

In some embodiments, a cell expressing a CAR described herein is a cell expressing a CD22 CAR (e.g., a cell expressing a CAR that binds to human CD22). In some embodiments, a cell expressing a CD22 CAR comprises an antigen-binding domain of WO2016164731 and WO2018067992, incorporated herein by reference. Exemplary CD22 binding sequences or CD22 CAR sequences are disclosed in, e.g., Tables 6A, 6B, 7A, 7B, 7C, 8A, 8B, 9A, 9B, 10A, and 10B of WO2016164731 and Tables 6-10 of WO2018067992. In some embodiments, the CD22 CAR sequence comprises a CDR, variable region, scFv, or full-length CD22 CAR sequence disclosed in WO2018067992 or WO2016164731.

In some embodiments, the CAR molecule comprises an antigen-binding domain that binds to CD22 (a CD22 CAR). In some embodiments, the antigen-binding domain targets human CD22. In some embodiments, the antigen-binding domain comprises a single-chain Fv sequence as described herein.

Sequences of the CAR for human CD22 are provided below. In some embodiments, the CAR for human CD22 is CAR22-65.

Sequence of scFv CAR for human CD22

EVQLQQSGPGLVKPSQTLSLTCAISGDSMLSNSDTWNWIRQSPSRGLEWLGRTYHRSTWYDDYASSVRGRVSINVDTSKNQYSLQLNAVTPEDTGVYYCARVRLQDGNSWSDAFDVWGQGTMVTVS SGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTQLTVL (SEQ ID NO: 285)

Human CD22 CAR heavy chain variable region

EVQLQQSGPGLVKPSQTLSLTCAISGDSMLSNSDTWNWIRQSPSRGLEWLGRTYHRSTWYDDYASSVRGRVSINVDTSKNQYSLQLNAVTPEDTGVYYCARVRLQDGNSWSDAFDVWGQGTMVTVSS (SEQ ID NO 286)

Human CD22 CAR light chain variable region

QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGTGTQLTVL (SEQ ID NO 287)

Table 16. CDRs of the CAR heavy chain variable domain for CD22 (CAR22-65) Candidate HCDR1 SEQ ID NO: HCDR2 SEQ ID NO: HCDR3 SEQ ID NO: CAR22-65 according to combination of definitions GDSMLSNSDTWN 288 RTYHRSTWYDDYASSVRG 290 VRLQDGNSWSDAFDV 291 CAR22-65 by Kabat SNSDTWN 289 RTYHRSTWYDDYASSVRG 290 VRLQDGNSWSDAFDV 291

Table 17. CAR light chain variable domain CDRs for CD22 (CAR22-65). The CDR LC sequences in this table have the same sequence according to the Kabat definition or a combination of definitions. Candidate LCDR1 SEQ ID NO: LCDR2 SEQ ID NO: LCDR3 SEQ ID NO: CAR22-65 according to combination of definitions TGTSSSDVGGYNYVS 95 DVSNRPS 96 SSYTSSSTLYV 97

In some embodiments, the antigen binding domain comprises CDR1 HC, CDR2 HC, and CDR3 HC with any of the heavy chain binding domain amino acid sequences listed in Table 16. In some embodiments, the antigen binding domain further comprises CDR1 LC, CDR2 LC, and CDR3 LC. In some embodiments, the antigen binding domain comprises the CDR1 LC, CDR2 LC, and CDR3 LC amino acid sequences listed in Table 17 .

In some embodiments, the antigen binding domain comprises one, two, or all of CDR1 LC, CDR2 LC, and CDR3 LC with any of the light chain binding domain amino acid sequences listed in Table 17 , and one, two, or all of CDR1 HC, CDR2 HC, and CDR3 HC with any of the heavy chain binding domain amino acid sequences listed in Table 16 .

In some embodiments, the CDRs are defined according to the Kabat numbering scheme, the Chothia numbering scheme, or a combination thereof.

The order in which the VL and VH domains are presented in the scFv may vary (i.e., in a VL-VH or VH-VL orientation), and any number of one, two, three, or four copies of "G4S" subunits (SEQ ID NO: 25), wherein each subunit comprises the sequence GGGGS (SEQ ID NO: 25) (e.g., (G4S) ₃ (SEQ ID NO: 28) or (G4S) ₄ (SEQ ID NO: 27)), may connect the variable domains to create an entire scFv domain. Alternatively, the CAR construct may comprise, for example, a linker comprising the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 43). Alternatively, the CAR construct may comprise, for example, a linker comprising the sequence LAEAAAK (SEQ ID NO: 308). In one embodiment, the CAR construct does not comprise a linker between the VL and VH domains.

All of these clones contained a Q/K residue alteration in the signaling domain of the costimulatory domain derived from the CD3 zeta chain.

CAR for EGFR

In some embodiments, a cell expressing a CAR described herein is a cell expressing a CAR for EGFR (e.g., a cell expressing a CAR that binds to human EGFR). In some embodiments, a cell expressing a CAR described herein is a cell expressing a CAR for EGFRvIII (e.g., a cell expressing a CAR that binds to human EGFRvIII). Exemplary CARs for EGFRvIII may comprise the sequences disclosed in WO2014/130657, such as Table 2 of WO2014/130657, incorporated herein by reference.

Exemplary EGFRvIII binding sequences or EGFR CAR sequences may comprise a CDR, variable region, scFv, or full-length CAR sequence from the EGFR CAR disclosed in WO2014/130657.

CAR for mesothelin

In some embodiments, a cell expressing a CAR described herein is a cell expressing a CAR for mesothelin (e.g., a cell expressing a CAR that binds to human mesothelin). Exemplary CARs for mesothelin may comprise the sequences disclosed in WO2015090230 and WO2017112741, such as Tables 2, 3, 4, and 5 of WO2017112741, incorporated herein by reference.

Other illustrative CARs

In other embodiments, the cells expressing the CAR may specifically bind to CD123, such as may comprise a CAR molecule (e.g., any of CAR1-CAR8) or an antigen binding domain according to Tables 1-2 of WO 2014/130635, incorporated herein by reference. The amino acid sequences of CAR molecules and antigen binding domains for CD123 (e.g., comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) and the nucleotide sequences encoding them are set forth in WO 2014/130635. In other embodiments, the cells expressing the CAR may specifically bind to CD123, such as may comprise a CAR molecule (e.g., any of CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32) or antigen binding domain thereof according to Tables 2, 6, and 9 of WO2016/028896, incorporated herein by reference. The amino acid sequences of CAR molecules and antigen binding domains for CD123 (e.g., comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) and the nucleotide sequences encoding them are set forth in WO2016/028896.

In some embodiments, the CAR molecule provides a CLL1 CAR described herein, such as a CLL1 CAR described in US2016/0051651A1, incorporated herein by reference. In some embodiments, the CLL1 CAR comprises an amino acid sequence or has a nucleotide sequence shown in US2016/0051651A1, incorporated herein by reference. In other embodiments, cells expressing the CAR may specifically bind to CLL-1, such as may comprise a CAR molecule or an antigen binding domain according to Table 2 of WO2016/014535, incorporated herein by reference. Amino acid sequences of CAR molecules and antigen-binding domains for CLL-1 (e.g. comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) and nucleotide sequences encoding them are disclosed in WO2016/014535.

In some embodiments, the CAR molecule provides a CD33 CAR described herein, such as a CD33 CAR described in US2016/0096892A1, incorporated herein by reference. In some embodiments, the CD33 CAR comprises an amino acid sequence or has a nucleotide sequence as shown in US2016/0096892A1, incorporated herein by reference. In other embodiments, cells expressing the CAR may specifically bind to CD33, such as may comprise a CAR molecule (e.g., any of CAR33-1 to CAR-33-9) or an antigen binding domain according to Table 2 or 9 of WO2016/014576, incorporated herein by reference. Amino acid sequences of CAR molecules and antigen-binding domains for CD33 (e.g. comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) and nucleotide sequences encoding them are disclosed in WO2016/014576.

In some embodiments, the antigen binding domain comprises one, two, three (e.g., all three) heavy chain CDRs - CDR1 HC, CDR2 HC, and CDR3 HC - from an antibody described herein (e.g., an antibody described in WO2015/142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014/0322212A1, US2016/0068601A1, US2016/0051651A1, US2016/0096892A1, US2014/0322275A1, or WO2015/090230, all incorporated herein by reference) and/or one, two, three (e.g., all three) light chain CDRs - CDR1 LC, CDR2 LC and CDR3 LC - from an antibody described herein (e.g., an antibody described in WO2015/142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014/0322212A1, US2016/0068601A1, US2016/0051651A1, US2016/0096892A1, US2014/0322275A1, or WO2015/090230, incorporated herein by reference). In some embodiments, the antigen-binding domain comprises a heavy chain variable region and/or a light chain variable region of an antibody as defined above.

In some embodiments, the antigen binding domain is an antigen binding domain described in WO2015/142675, US-2015-0283178-A1, US-2016-0046724-A1, US2014/0322212A1, US2016/0068601A1, US2016/0051651A1, US2016/0096892A1, US2014/0322275A1, or WO2015/090230, incorporated herein by reference.

In some embodiments, the antigen binding domain targets BCMA and is described in US-2016-0046724-A1. In some embodiments, the antigen binding domain targets CD19 and is described in US-2015-0283178-A1. In some embodiments, the antigen binding domain targets CD123 and is described in US2014/0322212A1, US2016/0068601A1. In some embodiments, the antigen binding domain targets CLL1 and is described in US2016/0051651A1. In some embodiments, the antigen binding domain targets CD33 and is described in US2016/0096892A1.

Exemplary target antigens that can be targeted by CAR expressing cells include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR-alpha 4, as described, for example, in WO2014/153270, WO2014/130635, WO2016/028896, WO2014/130657, WO2016/014576, WO2015/090230, WO2016/014565, WO2016/014535, and WO2016/025880, each of which is incorporated herein by reference in its entirety.

In other embodiments, the cells expressing the CAR may specifically bind to GFRalpha 4, for example, may comprise a CAR molecule or antigen binding domain according to Table 2 of WO2016/025880, incorporated herein by reference. The amino acid sequences of CAR molecules and antigen binding domains for GFRalpha 4 (e.g., comprising one, two, three VH CDRs and one, two, three VL CDRs according to Kabat or Chothia) and the nucleotide sequences encoding them are set forth in WO2016/025880.

In some embodiments, the antigen-binding domain of any of the CAR molecules described herein (e.g., for any of CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR-alpha 4) comprises one, two, three (e.g., all three) heavy chain CDRs - CDR1 HC, CDR2 HC, and CDR3 HC - from an antibody described above and/or one, two, three (e.g., all three) light chain CDRs - CDR1 LC, CDR2 LC, and CDR3 LC - from an antigen-binding domain described above. In some embodiments, the antigen-binding domain comprises a heavy chain variable region and/or a light chain variable region of an antibody described or referenced above.

In some embodiments, the antigen-binding domain comprises one, two, three (e.g., all three) heavy chain CDRs - CDR1 HC, CDR2 HC, and CDR3 HC - of an antibody as defined above and/or one, two, three (e.g., all three) light chain CDRs - CDR1 LC, CDR2 LC, and CDR3 LC - of an antibody as defined above. In some embodiments, the antigen-binding domain comprises a heavy chain variable region and/or a light chain variable region of an antibody as defined or described above.

In some embodiments, the tumor antigen is a tumor antigen as described in International Application WO2015/142675, filed March 13, 2015, incorporated herein by reference in its entirety. In some embodiments, the tumor antigen is selected from one or more of CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); B-cell maturation antigen (BCMA), which is a member of the TNF receptor family; Tn antigen ((Ag Tn) or (GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); orphan receptor 1, similar to receptor tyrosine kinase 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor alpha-2 subunit (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha subunit (IL-11Ra); prostate stem cell antigen (PSCA); serine protease 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis system antigen (Y); CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen 4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2/neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostasis; Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, multicatalytic protease) beta-type 9 subunit (LMP2); glycoprotein 100 (gp100); oncogenic fusion protein of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin A-type receptor 2 (EphA2); fucosyl-GM1; sialylated Lewis antigen (sLe) adhesion molecule; GM3 ganglioside (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker-related protein 7 (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); group C protein-coupled receptor 5 member D (GPRC5D); X chromosome open reading frame 61 protein encoded by CXORF61; CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific protein 1 (PLAC1); hexasaccharide portion of glycoceramide globoH (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); beta-3-adrenergic receptor (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen complex 6 locus K9 antigen (LY6K); olfactory receptor 51E2 (OR51E2); TCR-gamma alternative reading frame–encoded protein (TARP); Wilms tumor protein (WT1); cancer-testicular antigen 1 (NY-ESO-1); cancer-testicular antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); protein encoded by the ETS translocation variant 6, localized to the p-arm of chromosome 12 (ETV6-AML); sperm protein 17 (SPA17); X-antigen family member 1A (XAGE1); angiopoietin-binding receptor-cell surface 2 (TIE-2); melanoma cancer-testicular antigen 1 (MAD-CT-1); melanoma cancer-testicular antigen 2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); mutant p53; prostein; survivin; telomerase; Prostate carcinoma tumor antigen 1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1); mutant rat sarcoma antigen (Ras); human telomerase reverse transcriptase (hTERT); breakpoint translocation sarcoma antigen; melanoma inhibitor of apoptosis (ML-IAP); ERG (fusion product of the transmembrane serine protease 2 gene (TMPRSS2) and ETS); N-acetylglucosaminyltransferase V (NA17); Pax paired-box protein 3 (PAX3); androgen receptor; cyclin B1; neuroblastoma homolog of avian myelocytomatosis virus oncogene v-myc (MYCN); Ras homolog family member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); Zinc finger protein CCCTC-binding factor-like protein (BORIS or brother regulator of imprinted sites), Squamous cell carcinoma antigen recognized by T cells 3 (SART3); Pax paired box domain protein 5 (PAX5); Proacrosin-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); Kinase A anchor protein 4 (AKAP-4); Synovial sarcoma X-linked breakpoint antigen 2 (SSX2); Receptor for advanced glycation end products (RAGE-1); Renal ubiquitin protein 1 (RU1); Renal ubiquitin protein 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mutant variant of hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA Fc fragment receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like protein f family member (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); hormone receptor mucin-like protein containing EGF-like module 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like antigen 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

In some embodiments, the antigen-binding domain comprises one, two, three (e.g., all three) heavy chain CDRs - CDR1 HC, CDR2 HC, and CDR3 HC - of an antibody as defined above and/or one, two, three (e.g., all three) light chain CDRs - CDR1 LC, CDR2 LC, and CDR3 LC - of an antibody as defined above. In some embodiments, the antigen-binding domain comprises a heavy chain variable region and/or a light chain variable region of an antibody as defined or described above.

In some embodiments, the tumor antigen binding domain is a fragment, such as a single chain variable fragment (scFv). In some embodiments, the binding domain for a cancer associated antigen described herein is an Fv, a Fab (Fab')2, or a bifunctional (e.g., bispecific) chimeric antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In some embodiments, the antibodies and fragments thereof of the present invention bind to a protein that is a cancer associated antigen described herein with wild-type or enhanced affinity.

In some cases, scFv can be prepared according to a method known in the art (see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be prepared by linking the VH and VL regions to each other using flexible polypeptide linkers. scFv molecules contain a linker (e.g., a Ser-Gly linker) with optimized length and/or amino acid composition. The length of the linker can greatly influence the folding and interaction of the variable regions of scFv. Indeed, if a short polypeptide linker (e.g. 5-10 amino acids long) is used, intrachain folding is prevented. Interchain folding is also necessary to bring the two variable regions together to form a functional epitope-binding site. For examples of linker orientation and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference.

The scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In some embodiments, the linker sequence comprises groups of glycine and serine repeats, such as (Gly ₄ Ser)n, wherein n is a positive integer that is equal to or greater than 1 (SEQ ID NO: 25). In some embodiments, the linker may be (Gly ₄ Ser) ₄ (SEQ ID NO: 27) or (Gly ₄ Ser) ₃ (SEQ ID NO: 28). By changing the length of the linker, activity may be maintained or enhanced, resulting in improved performance in activity assays.

In some embodiments, the antigen-binding domain is a T cell receptor ("TCR") or a fragment thereof, e.g., a single-chain TCR (scTCR). Methods for producing such TCRs are known in the art. See, e.g., Willemsen RA et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (the references are incorporated herein in their entireties). For example, an scTCR can be engineered that comprises the Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very applicable for a cancer-associated target, which is itself intracellular, but a fragment of such an antigen (peptide) is presented on the surface of cancer cells using MHC.

Transmembrane domain

With respect to the transmembrane domain, in various embodiments, the CAR can be designed to comprise a transmembrane domain attached to an extracellular domain of the CAR. The transmembrane domain can comprise one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids related to the extracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and up to 15 amino acids of the extracellular region), and/or one or more additional amino acids related to the intracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and up to 15 amino acids of the intracellular region). In some embodiments, the transmembrane domain is a domain associated with one of the remaining domains of the CAR used. In some cases, the transmembrane domain may be selected or modified by an amino acid substitution to avoid association of such domains with transmembrane domains of the same or other surface membrane proteins, for example, to minimize interactions with other elements of the receptor complex. In some embodiments, the transmembrane domain is capable of homodimerization with another CAR on the surface of a cell expressing the CAR, for example, a CAR-T cell. In some embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted in a manner that minimizes interactions with the binding domains of a native binding partner present in the same cell expressing the CAR, for example, a CAR-T.

The transmembrane domain may be obtained from a natural or recombinant source. If the source is natural, the domain may be obtained from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain is capable of transmitting a signal to the intracellular domain(s) whenever the CAR has bound to a target. A transmembrane domain particularly useful in the present invention may comprise at least a transmembrane region(s) such as the alpha, beta or zeta chains of a T cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8 (e.g., CD8alpha, CD8beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain may comprise at least the transmembrane region(s) of a costimulatory molecule, such as an MHC class I molecule, TNF protein receptors, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activating receptors, BTLA, Toll-like receptor ligand, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and a ligand that specifically binds to CD83.

In some cases, the transmembrane domain may be attached to an extracellular region of the CAR, such as an antigen-binding domain of the CAR, via a hinge region, such as a hinge region from a human protein. For example, in some embodiments, the hinge region may be a human Ig (immunoglobulin) hinge region, such as an IgG4 hinge region or a CD8a hinge region. In some embodiments, the hinge region or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 2. In some embodiments, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 6.

In some embodiments, the hinge region or spacer comprises an IgG4 hinge region. For example, in some embodiments, the hinge region or spacer comprises the hinge region of SEQ ID NO: 3. In some embodiments, the hinge region or spacer comprises a hinge region encoded by the nucleotide sequence of SEQ ID NO: 14.

In some embodiments, the hinge region or spacer comprises an IgD hinge region. For example, in some embodiments, the hinge region or spacer comprises a hinge region with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the hinge region or spacer comprises a hinge region encoded by the nucleotide sequence of SEQ ID NO: 15.

In some embodiments, the transmembrane domain may be recombinant, in which case it will contain primarily hydrophobic residues such as leucine and valine. In some embodiments, at each end of the recombinant transmembrane domain, there may be a triplet of phenylalanine, tryptophan, and valine.

A short oligo- or polypeptide linker of 2 to 10 amino acids in length may optionally form a link between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet is a particularly suitable linker. For example, in some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the linker is encoded by the nucleotide sequence of SEQ ID NO: 16.

In some embodiments, the hinge region or spacer comprises a KIR2DS2 hinge region.

Cytoplasmic domain

The cytoplasmic domain or region of the CAR of the present invention comprises an intracellular signaling domain. The intracellular signaling domain is typically responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced.

Examples of intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signaling upon engagement with an antigen receptor, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capabilities.

It is known that signals generated by the TCR alone are insufficient for full T cell activation and that a secondary and/or costimulatory signal is also required. Thus, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to transmit a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., costimulatory domain).

The primary signaling domain regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

Examples of ITAM-containing primary intracellular signaling domains that are particularly useful in the present invention include domains from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d. In some embodiments, a CAR of the present invention comprises an intracellular signaling domain, such as a CD3 zeta primary signaling domain.

In some embodiments, the primary signaling domain comprises a modified ITAM domain, such as a mutant ITAM domain that has altered (e.g., increased or decreased) activity compared to a native ITAM domain. In some embodiments, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, such as an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In some embodiments, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

Further examples of primary intracellular signaling domain-containing molecules that are particularly useful in the present invention include DAP10, DAP12 and CD32 molecules.

The intracellular signaling domain of the CAR may comprise a primary signaling domain, such as a CD3 zeta signaling domain, alone or in combination with any other desired intracellular signaling domain(s) useful as part of the CAR of the present invention. For example, the intracellular signaling domain of the CAR may comprise a primary signaling domain, such as a portion of the CD3 zeta chain, and a costimulatory signaling domain. The costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligands, that is necessary for an effective lymphocyte response to an antigen. Examples of such a molecule include MHC class I molecule, TNF protein receptors, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), NK cell activating receptors, BTLA, Toll-like receptor ligand, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and a ligand that specifically binds to CD83, etc. For example, costimulation with CD27 has been demonstrated to enhance the expansion, effector function and survival of human CAR T cells in vitro and enhance the persistence and antitumor activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706). The intracellular signal sequences in the cytoplasmic portion of the CAR of the present invention may be linked to each other in a random or fixed order. A short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may optionally form a link between the intracellular signal sequences. In some embodiments, a glycine-serine doublet may be used as a suitable linker. In some embodiments, a single amino acid, such as alanine, glycine, may be used as a suitable linker.

In some embodiments, the intracellular signaling domain is designed to comprise two or more, such as 2, 3, 4, 5, or more, costimulatory signaling domains. In some embodiments, the two or more, such as 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, such as a linker molecule described herein. In some embodiments, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

In some embodiments, the intracellular signaling domain is designed to comprise a CD3-zeta signaling domain and a CD28 signaling domain. In some embodiments, the intracellular signaling domain is designed to comprise a CD3-zeta signaling domain and a 4-1BB signaling domain. In some embodiments, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 7. In some embodiments, the CD3-zeta signaling domain is the signaling domain of SEQ ID NO: 9 (mutant CD3-zeta) or SEQ ID NO: 10 (wild-type human CD3-zeta).

In some embodiments, the intracellular signaling domain is designed to comprise a CD3-zeta signaling domain and a CD27 signaling domain. In some embodiments, the CD27 signaling domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CD27 signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 19.

In some embodiments, the intracellular domain is designed to comprise a CD3-zeta signaling domain and a CD28 signaling domain. In some embodiments, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the CD28 signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 37.

In some embodiments, the intracellular domain is designed to comprise a CD3-zeta signaling domain and an ICOS signaling domain. In some embodiments, the ICOS signaling domain comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the ICOS signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 39.

CAR Configurations

Double CARs

In one embodiment, an immune cell (e.g., a T cell or an NK cell) expresses two CARs, such as a first CAR that binds to a first antigen and a second CAR that binds to a second antigen. In one embodiment, the first antigen and the second antigen are different. In one embodiment, the first or second antigen is selected from an antigen expressed on B cells, an antigen expressed on acute myeloid leukemia cells, or an antigen on solid tumor cells. In one embodiment, the first or second antigen is selected from CD10, CD19, CD20, CD22, CD34, CD123, BCMA, FLT-3, ROR1, CD79b, CD179b, CD79a, CD34, CLL-1, folate receptor beta, FLT3, EGFRvIII, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (eg, ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mutant hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC.

In one embodiment, the first antigen is CD19. In one embodiment, the second antigen is not CD19. In one embodiment, the second antigen is an antigen disclosed herein that is not CD19.

In one embodiment, the first antigen is BCMA. In one embodiment, the second antigen is not BCMA. In one embodiment, the second antigen is an antigen disclosed herein that is not BCMA. In one embodiment, the second antigen is selected from an antigen expressed on B cells, an antigen expressed on acute myeloid leukemia cells, or an antigen on solid tumor cells. In one embodiment, the second antigen is selected from CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, CD79a, CD34, CLL-1, folate receptor beta, FLT3, EGFRvIII, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (eg, ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mutant variant hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC. In one embodiment, the first antigen is BCMA and the second antigen is CD19.

In one embodiment, the first CAR is encoded by a first nucleic acid sequence. In one embodiment, the second CAR is encoded by a second nucleic acid sequence. In one embodiment, the first and second nucleic acid sequences are located on a single nucleic acid molecule. In one embodiment, the first and second nucleic acid sequences are located on separate nucleic acid molecules. In one embodiment, the nucleic acid molecule or molecules are DNA or RNA molecules. In some embodiments, the first and second nucleic acid sequences are in the same orientation, such that transcription of the first and second nucleic acid sequences occurs in the same direction. In some embodiments, the first and second nucleic acid sequences are in different orientations. In some embodiments, a single promoter controls expression of the first and second nucleic acid sequences. In some embodiments, a nucleic acid encoding a protease cleavage site (such as a T2A, P2A, E2A, or F2A cleavage site) is located between the first and second nucleic acid sequences. In some embodiments, the protease cleavage site is positioned such that the cell can express a fusion protein comprising a first CAR and a second CAR, and the fusion protein is subsequently processed by proteolytic cleavage to form two peptides. In some embodiments, the first nucleic acid sequence is located upstream of the second nucleic acid sequence, or the second nucleic acid sequence is located upstream of the first nucleic acid sequence. In some embodiments, the first promoter controls expression of the first nucleic acid sequence, and the second promoter controls expression of the second nucleic acid sequence. In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the nucleic acid molecule comprises a viral packaging element. In some embodiments, the immune cell may comprise a protease (e.g., an endogenous or exogenous protease) that cleaves a T2A, P2A, E2A, or F2A cleavage site.

In one embodiment, the first CAR comprises a first antigen-binding domain and the second CAR comprises a second antigen-binding domain. In one embodiment, the first or second antigen-binding domain comprises a CDR, VH, VL, or scFv disclosed herein, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In one embodiment, the first or second antigen-binding domain comprises a CDR, VH, VL, or scFv of an antigen-binding domain for BCMA disclosed herein (e.g., an amino acid sequence disclosed in Tables 3-15, 19, 20, 22, and 26, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In one embodiment, the first or second antigen-binding domain comprises a CDR, VH, VL, or scFv of an antigen-binding domain for CD19 disclosed herein (e.g., an amino acid sequence disclosed in Tables 2, 19, and 22, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

In one embodiment, the first antigen is BCMA and the second antigen is CD19. In one embodiment, the immune cell (e.g., a T cell or NK cell) expresses a CAR for BCMA, such as a CAR for BCMA described herein, and a CAR for CD19, such as a CAR for CD19 described herein. In one embodiment, the immune cell (e.g., a T cell or NK cell) comprises a first nucleic acid sequence encoding a CAR for BCMA, such as a CAR for BCMA described herein, and a second nucleic acid sequence encoding a CAR for CD19, such as a CAR for CD19 described herein. Table 19 shows exemplary amino acid sequences and nucleic acid sequences of dual CAR constructs.

Table 19. Illustrative BCMA/CD19 dual CAR designs and their components Dual CAR designs for BCMA/CD19 Identification Protein sequence DNA sequence (5'-3') Signal peptide
(in the CAR arm for BCMA) MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) Atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggccc (SEQ ID NO: 199) ScFv R1G5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK (SEQ ID NO: 80) Gaagtgcagttgctggagtcaggcggaggactggtgcagcccggaggatcgcttcgcttgagctgcgcagcctcaggctttaccttctcctcc tacgccatgtcctgggtcagacaggctcccgggaagggactggaatgggtgtccgccattagcggttccggcggaagcacttactatgccgac tctgtgaagggccgcttcactatctcccgggacaactccaagaacaccctgtatctccaaatgaattccctgagggccgaagataccgcggtgtactactgcgctagacgggagtggtggggagaaagctggctgttcgactactggggacagggcactctcgtgactgtgtcctccggtggtggt ggatcggggggtggtggttcggggcggaggaggatctggaggaggagggtcggacattcaaatgactcagtccccgtcctccctctccgcctccgtggggagatcgcgtcacgatcacgtgcaggccagccagagcatctccagctacctgaactggtaccagcagaagccagggaaggcaccgaag ctcctgatctacgccgctagctcgctgcagtccggcgtcccttcacggttctcgggatcgggctcaggcaccgacttcaccctgaccattagcagcctgcagccggaggacttcgcgacatactactgtcagcagtcatactccacccctctgaccttcggccaagggaccaaagtggagatcaag (SEQ ID NO: 81) ScFvPI61 QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSG GGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL (SEQ ID NO: 105) Caagtgcagctgcaggaatccggtggcggagtcgtgcagcctggaaggagcctgagactctcatgcgccgcgtcagggttcaccttttcctcc tacgggatgcattgggtcagacaggcccccggaaagggactcgaatgggtggctgtgatcagctacgacggctccaacaagtactacgccgac tccgtgaaaggccggttcactatctcccgggacaactccaagaacacgctgtatctgcaaatgaattcactgcgcgcggaggataccgctgtgtactactgcggtggctccggttacgccctgcacgatgactattacggccttgacgtctggggccagggaaccctcgtgactgtgtccagcggtg gaggaggttcgggcggaggaggatcaggagggggtggatcgcagagcgcactgactcagccggcatccgtgtccggtagccccggacagtcgattaccatctcctgtaccggcacctcctccgacgtgggagggtacaactacgtgtcgtggtaccagcagcacccaggaaaggcccctaagttgat gatctacgatgtgtcaaaccgcccgtctggagtctccaaccggttctccggctccaagtccggcaacaccgccagcctgaccattagcgggctgcaagccgaggatgaggccgactactactgctcgagctacacatcctcgagcaccctctacgtgttcggctcggggactaaggtcaccgtgctg (SEQ ID NO: 106) ScFv R1B6 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK (SEQ ID NO: 64) Gaagtgcagttgctggagtcaggcggaggactggtgcagcccggaggatcgcttcgcttgagctgcgcagcctcaggctttaccttctcctcc tacgccatgtcctgggtcagacaggctcccgggaagggactggaatgggtgtccgccattagcggttccggcggaagcacttactatgccgact ctgtgaagggccgcttcactatctcccgggacaactccaagaacaccctgtatctccaaatgaattccctgagggccgaagataccgcggtgtactactgcgctagacgggagtgggtgccctacgatgtcagctggtacttcgactactggggacagggcactctcgtgactgtgtcctccggtggt ggtggatcggggggtggtggttcggggcggaggaggatctggaggaggagggtcggacattcaaatgactcagtccccgtcctccctctccgcctccgtggggagatcgcgtcacgatcacgtgcaggggccagccagagcatctccagctacctgaactggtaccagcagaagccagggaaggcaccga agctcctgatctacgccgctagctcgctgcagtccggcgtcccttcacggttctcgggatcgggctcaggcaccgacttcaccctgaccattagcagcctgcagccggaggacttcgcgacatactactgtcagcagtcatactccacccctctgaccttcggccaagggaccaaagtggagatcaag (SEQ ID NO: 65) ScFv duBCMA.4 EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGG GGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK (SEQ ID NO: 200) Gaagtgcaattggtggaatcagggggaggacttgtgcagcctggaggatcgctgagactgtcatgtgccgtgtccggctttgccctgtccaaccacgggatgtcctgggtccgccgcgcgcctggaaagggcctcgaatgggtgtcgggtattgtgtacagcggtagcacctactatgc cgcatccgtgaaggggagattcaccatcagccgggacaactccaggaacactctgtacctccaaatgaattcgctgaggccagaggacactgccatctactactgctccgcgcatggcggagagtccgacgtctggggacaggggaccaccgtgaccgtgtctagcgcgtccggcggag gcggcagcgggggtggtggttcagggggcggcggatcggacatccagctcacccagtccccgagctcgctgtccgcctccgtggggagatcgggtcaccatcacgtgccgcgccagccagtcgatttcctcctacctgaactggtaccaacagaagcccggaaaagccccgaagcttctc atctacgccgcctcgagcctgcagtcaggagtgccctcacggttctccggctccggttccggtactgatttcaccctgaccatttcctccctgcaaccggaggacttcgctacttactactgccagcagtcgtactccaccccctacactttcggacaaggcaccaaggtcgaaatcaag (SEQ ID NO: 201) Hinge region and transmembrane domain
(in the CAR arm for BCMA) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 202) Accactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccg tgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgt (SEQ ID NO: 203) Hinge area
(in the CAR arm for BCMA) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 2) Accactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgat (SEQ ID NO: 337) Transmembrane domain
(in the CAR arm for BCMA) IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 6) Atctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgt (SEQ ID NO: 338) 4-1BB
(in the CAR arm for BCMA) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7) Aagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggagggaaggcggctgcgaactg (SEQ ID NO: 204) CD3-zeta
(in the CAR arm for BCMA) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) Cgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagag ggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 205) Linker GSG (SEQ ID NO: 206) Ggaagcgga (SEQ ID NO: 207) P2A sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 208) Gctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacct (SEQ ID NO: 209) Signal peptide
(in the CAR arm for CD19) MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg (SEQ ID NO: 210) ScFv duCD19.1 EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGG SQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 211) Gaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgcca ggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggagga agccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacc aatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagc (SEQ ID NO: 212) Hinge region and transmembrane domain
(in CAR arm for CD19) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 202) Accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcag tgcacacgaggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 213) Hinge area
(in CAR arm for CD19) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 2) Accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgaggggctggacttcgcctgtgat (SEQ ID NO: 13) Transmembrane domain
(in CAR arm for CD19) IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 6) Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 17) 4-1BB
(in CAR arm for CD19) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7) Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (SEQ ID NO: 18) CD3-zeta
(in CAR arm for CD19) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) Agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaa ggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 21) R1G5-P2A-duCD19.1 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGE SWLFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPLTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFS LLKQAGDVEENPGPMALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQ GNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVY YCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 214) atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaagtgcagttgctggagtcaggcggagga ctggtgcagcccggaggatcgcttcgcttgagctgcgcagcctcaggctttaccttctcctcctacgccatgtcctgggtcagacaggctcccg ggaagggactggaatgggtgtccgccattagcggttccggcggaagcacttactatgccgactctgtgaagggccgcttcactatctcccgggacaactccaagaacaccctgtatctccaaatgaattccctgagggccgaagataccgcggtgtactactgcgctagacgggagtggtggggagaa agctggctgttcgactactggggacagggcactctcgtgactgtgtcctccggtggtggtggatcggggggtggtggttcgggcggaggagga tctggaggaggagggtcggacattcaaatgactcagtccccgtcctccctctccgcctccgtggggagatcgcgtcacgatcacgtgcagggcca gccagagcatctccagctacctgaactggtaccagcagaagccagggaaggcaccgaagctcctgatctacgccgctagctcgctgcagtccggcgtcccttcacggttctcgggatcgggctcaggcaccgacttcaccctgaccattagcagcctgcagccggaggacttcgcgacatactactgt cagcagtcatactccacccctctgaccttcggccaagggaccaaagtggagatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcct gcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggagggaggaa ggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagaggggcc tgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggggaagcggagctactaacttcagc ctgctgaagcaggctggagacgtggaggagaaccctggacctatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaag acatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaa gggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccg attacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtac tattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgc acacgaggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagc tgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccga gaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 215) PI61-P2A-duCD19.1 MALPVTALLLPLALLLHAARPQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALH DDYYGLDVWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCS SYTSSSTLYVFGSGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNF SLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQ QGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAV YYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 216) atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggccccaagtgcagctgcaggaatccggtggcgga gtcgtgcagcctggaaggagcctgagactctcatgcgccgcgtcagggttcaccttttcctcctacgggatgcattgggtcagacaggcccccg gaaagggactcgaatgggtggctgtgatcagctacgacggctccaacaagtactacgccgactccgtgaaaggccggttcactatctcccgggacaactccaagaacacgctgtatctgcaaatgaattcactgcgcgcggaggataccgctgtgtactactgcggtggctccggttacgccctgcac gatgactattacggccttgacgtctggggccagggaaccctcgtgactgtgtccagcggtggaggaggttcgggcggaggaggatcaggagggggtggatcgcagagcgcactgactcagccggcatccgtgtccggtagccccggacagtcgattaccatctcctgtaccggcacctcctccgacg tgggagggtacaactacgtgtcgtggtaccagcagcacccaggaaaggcccctaagttgatgatctacgatgtgtcaaaccgcccgtctggagtctccaaccggttctccggctccaagtccggcaacaccgccagcctgaccattagcgggctgcaagccgaggatgaggccgactactactgctcg agctacacatcctcgagcaccctctacgtgttcggctcggggactaaggtcaccgtgctgaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcg cctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggag gaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagg cctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggggaagcggagctactaacttca gcctgctgaagcaggctggagacgtggaggagaaccctggacctatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctccca agacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagc aagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctcccc gattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgta ctattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg cacacgaggggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtag ctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccga gaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 217) R1B6-P2A-duCD19.1 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPY DVSWYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPLTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNF SLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQ QGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAV YYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 218) atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaagtgcagttgctggagtcaggcggagga ctggtgcagcccggaggatcgcttcgcttgagctgcgcagcctcaggctttaccttctcctcctacgccatgtcctgggtcagacaggctcccg ggaagggactggaatgggtgtccgccattagcggttccggcggaagcacttactatgccgactctgtgaagggccgcttcactatctcccgggacaactccaagaacaccctgtatctccaaatgaattccctgagggccgaagataccgcggtgtactactgcgctagacgggagtgggtgccctac gatgtcagctggtacttcgactactggggacagggcactctcgtgactgtgtcctccggtggtggtggatcggggggtggtggttcgggcggag gaggatctggaggaggagggtcggacattcaaatgactcagtccccgtcctccctctccgcctccgtggggagatcgcgtcacgatcacgtgcag ggccagccagagcatctccagctacctgaactggtaccagcagaagccagggaaggcaccgaagctcctgatctacgccgctagctcgctgcagtccggcgtcccttcacggttctcgggatcgggctcaggcaccgacttcaccctgaccattagcagcctgcagccggaggacttcgcgacatact actgtcagcagtcatactccacccctctgaccttcggccaagggaccaaagtggagatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttc gcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggagga ggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagg gcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggggaagcggagctactaacttc agcctgctgaagcaggctggagacgtggaggagaaccctggacctatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctccc aagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcag caagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccc cgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgt actattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg cacacgaggggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtag ctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccga gaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 219) duBCMA.4-P2A-duCD19.1 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGES DVWGQGTTVTVSSASGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PYTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQ AGDVEENPGPMALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNT LPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYC AKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 220) atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaagtgcaattggtggaatcagggggaggacttgtgcagcctggaggatcgctgagactgtcatgtgccgtgtccggctttgccctgtccaaccacgggatgtcctgggtccgccgcgcgcc tggaaagggcctcgaatgggtgtcgggtattgtgtacagcggtagcacctactatgccgcatccgtgaaggggagattcaccatcagccgggacaactccaggaacactctgtacctccaaatgaattcgctgaggccagaggacactgccatctactactgctccgcgcatggcggagagtccga cgtctggggacaggggaccaccgtgaccgtgtctagcgcgtccggcggaggcggcagcgggggtggtggttcaggggggcggcggatcggacat ccagctcacccagtccccgagctcgctgtccgcctccgtgggagatcgggtcaccatcacgtgccgcgccagccagtcgatttcctcctacct gaactggtaccaacagaagcccggaaaagccccgaagcttctcatctacgccgcctcgagcctgcagtcaggagtgccctcacggttctccggctccggttccggtactgatttcaccctgaccatttcctccctgcaaccggaggacttcgctacttactactgccagcagtcgtactccacccc ctacactttcggacaaggcaccaaggtcgaaatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttggg cccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaaggcggctgcgaactgc gcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctcc aaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcggggaagcggagctactaacttcagcctgctgaagcagg ctggagacgtggaggagaaccctggacctatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaa taccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacacc ctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggg gtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgc gctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacg aggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgc cgatttccagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgaga aggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 221) duCD19.1-P2A-duBCMA.4 MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGT KLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGS YAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSL LKQAGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDT AIYYCSAHGGESDVWGQGTTVTVSSASGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSYSTPYTFGQGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 222) atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccgg acaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaa gctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaa gggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagcta cgcaatggattactggggacagggtactctggtcaccgtgtccagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatct acatctgggcgcccttggccggggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggat gtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtaca atgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcggaagcggagctactaacttcagcctgc tgaagcaggctggagacgtggaggagaaccctggacctatggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaagtgcaattggtggaatcagggggaggacttgtgcagcctggaggatcgctgagactgtcatgtgccgtgtccggctttgcc ctgtccaaccacgggatgtcctgggtccgccgcgcgcctggaaagggcctcgaatgggtgtcgggtattgtgtacagcggtagcacctactatgccgcatccgtgaaggggagattcaccatcagccgggacaactccaggaacactctgtacctccaaatgaattcgctgaggccagaggacact gccatctactactgctccgcgcatggcggagagtccgacgtctggggacaggggaccaccgtgaccgtgtctagcgcgtccggcggaggcggcagcgggggtggtggttcaggggggcggcggatcggacatccagctcacccagtccccgagctcgctgtccgcctccgtgggagatcgggtcacc atcacgtgccgcgccagccagtcgatttcctcctacctgaactggtaccaacagaagcccggaaaagccccgaagcttctcatctacgccgcctcgagcctgcagtcaggagtgccctcacggttctccggctccggttccggtactgatttcaccctgaccatttcctccctgcaaccggaggac ttcgctacttactactgccagcagtcgtactccaccccctacactttcggacaaggcaccaaggtcgaaatcaagaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacc cggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgc cggttcccagaggaggagaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctaccagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgc agaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 223) Predicted proteins obtained from
R1G5-P2A-duCD19.1 CAR Shoulder for BCMA:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVE IKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG (SEQ ID NO: 224)
CAR Shoulder for CD19:
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 225) Predicted proteins obtained from
PI61-P2A-duCD19.1 CAR Shoulder for BCMA:
QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS GGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKV TVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG (SEQ ID NO: 226)
CAR Shoulder for CD19:
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 225) Predicted proteins obtained from
R1B6-P2A-duCD19.1 CAR Shoulder for BCMA:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKV EIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG (SEQ ID NO: 227)
CAR Shoulder for CD19:
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 225) Predicted proteins obtained from
duBCMA.4-P2A-duCD19.1 CAR Shoulder for BCMA:
EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGGG GSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIKTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG (SEQ ID NO: 228)
CAR Shoulder for CD19:
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 225) Predicted proteins obtained from
duCD19.1-P2A-duBCMA.4 CAR Shoulder for CD19:
EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGG SQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG (SEQ ID NO: 229)
CAR Shoulder for BCMA:
EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS ASGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFG QGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 230)

Table 22. Amino acid sequences of illustrative components of dual CARs SEQ ID NO Name/
description Subsequence PI61 SEQ ID NO:86 HCDR1 (Kabat) SYGMH SEQ ID NO:87 HCDR2 (Kabat) VISYDGSNKYYADSVKG SEQ ID NO:88 HCDR3 (Kabat) SGYALHDDYYGLDV SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:89 HCDR2 (Chothia) SYDGSN SEQ ID NO:88 HCDR3 (Chothia) SGYALHDDYYGLDV SEQ ID NO:90 HCDR1 (IMGT) GFTFSSYG SEQ ID NO:91 HCDR2 (IMGT) ISYDGSNK SEQ ID NO:92 HCDR3 (IMGT) GGSGYALHDDYYGLDV SEQ ID NO:93 VH QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO:94 DNA VH CAAGTGCAGCTGCAGGAATCCGGTGGCGGAGTCGTGCAGCCTGGAAGGAGCCTGAGACTCTCATGCGCCGCGTCAGGGTTCACCTTTTCCTCCTACGGGATGCATTGGGTCAGACAGGCCCCCGGAAAGGGACTCGAATGGGTGGCTGTGATCAGCTACGACGGCTCCAACAAGTACTACGCCG ACTCCGTGAAAGGCCGGTTCACTATCTCCCGGGACAACTCCAAGAACACGCTGTATCTGCAAATGAATTCACTGCGCGCGGAGGATACCGCTGTGTACTACTGCGGTGGCTCCGGTTACGCCCTGCACGATGACTATTACGGCCTTGACGTCTGGGGCCAGGGAACCCTCGTGACTGTGTCCAGC SEQ ID NO:95 LCDR1 (Kabat) TGTSSSDVGGYNYVS SEQ ID NO:96 LCDR2 (Kabat) DVSNRPS SEQ ID NO:97 LCDR3 (Kabat) SSYTSSSTLYV SEQ ID NO:98 LCDR1 (Chothia) TSSDVGGYNY SEQ ID NO:99 LCDR2 (Chothia) DVS SEQ ID NO: 100 LCDR3 (Chothia) YTSSSTYLE SEQ ID NO: 101 LCDR1 (IMGT) SSDVGGYNY SEQ ID NO:99 LCDR2 (IMGT) DVS SEQ ID NO:97 LCDR3 (IMGT) SSYTSSSTLYV SEQ ID NO: 102 VL QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO: 103 DNA VL CAGAGCGCACTGACTCAGCCGGCATCCGTGTCCGGTAGCCCCGGACAGTCGATTACCATCTCCTGTACCGGCACCTCCTCCGACGTGGGAGGGTACAACTACGTGTCGTGGTACCAGCAGCACCCAGGAAAGGCCCCTAAGTTGATGATCTACGATGTGTCAAACC GCCCGTCTGGAGTCTCCAACCGGTTCTCCGGCTCCAAGTCCGGCAACACCGCCAGCCTGACCATTAGCGGGCTGCAAGCCGAGGATGAGGCCGACTACTACTGCTCGAGCTACACATCCTCGAGCACCCTCTACGTGTTCGGCTCGGGGACTAAGGTCACCGTGCTG SEQ ID NO: 104 Linker GGGGSGGGGSGGGGS SEQ ID NO: 105 scFv (VH-linker-VL) QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSG GGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO: 106 DNA scFv CAAGTGCAGCTGCAGGAATCCGGTGGCGGAGTCGTGCAGCCTGGAAGGAGCCTGAGACTCTCATGCGCCGCGTCAGGGTTCACCTTTTCCTCCTACGGGATGCATTGGGTCAGACAGGCCCCCGGAAAGGGACTCGAATGGGTGGCTGTGATCAGCTACGACGGCTCCAACAAGTACTACGCCGAC TCCGTGAAAGGCCGGTTCACTATCTCCCGGGACAACTCCAAGAACACGCTGTATCTGCAAATGAATTCACTGCGCGCGGAGGATACCGCTGTGTACTACTGCGGTGGCTCCGGTTACGCCCTGCACGATGACTATTACGGCCTTGACGTCTGGGGCCAGGGAACCCTCGTGACTGTGTCCAGCGGTG GAGGAGGTTCGGGCGGAGGAGGATCAGGAGGGGGTGGATCGCAGAGCGCACTGACTCAGCCGGCATCCGTGTCCGGTAGCCCCGGACAGTCGATTACCATCTCCTGTACCGGCACCTCCTCCGACGTGGGAGGGTACAACTACGTGTCGTGGTACCAGCAGCACCCAGGAAAGGCCCCTAAGTTGAT GATCTACGATGTGTCAAACCGCCCGTCTGGAGTCTCCAACCGGTTCTCCGGCTCCAAGTCCGGCAACACCGCCAGCCTGACCATTAGCGGGCTGCAAGCCGAGGATGAGGCCGACTACTACTGCTCGAGCTACACATCCTCGAGCACCCTCTACGTGTTCGGCTCGGGGACTAAGGTCACCGTGCTG R1B6 SEQ ID NO:44 HCDR1 (Kabat) SYAMS SEQ ID NO:45 HCDR2 (Kabat) AISGSGGSTYYADSVKG SEQ ID NO:46 HCDR3 (Kabat) REWVPYDVSWYFDY SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:48 HCDR2 (Chothia) SGSGGS SEQ ID NO:46 HCDR3 (Chothia) REWVPYDVSWYFDY SEQ ID NO:49 HCDR1 (IMGT) GFTFSSYA SEQ ID NO:50 HCDR2 (IMGT) ISGSGGST SEQ ID NO:51 HCDR3 (IMGT) ARREWVPYDVSWYFDY SEQ ID NO:52 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTLVTVSS SEQ ID NO:53 DNA VH GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCG ACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGGTGCCCTACGATGTCAGCTGGTACTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:56 LCDR3 (Kabat) QQSYSTPLT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:59 LCDR3 (Chothia) SYSTPL SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:56 LCDR3 (IMGT) QQSYSTPLT SEQ ID NO:61 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:62 DNA VL GACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGC TGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:64 scFv (VH-linker-VL) EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWVPYDVSWYFDYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:65 DNA scFv GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCGACT CTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGGTGCCCTACGATGTCAGCTGGTACTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCCGGTGGT GGTGGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGA AGCTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG R1G5 SEQ ID NO:44 HCDR1 (Kabat) SYAMS SEQ ID NO:45 HCDR2 (Kabat) AISGSGGSTYYADSVKG SEQ ID NO:76 HCDR3 (Kabat) REWWGESWLFDY SEQ ID NO:47 HCDR1 (Chothia) GFTFSSY SEQ ID NO:48 HCDR2 (Chothia) SGSGGS SEQ ID NO:76 HCDR3 (Chothia) REWWGESWLFDY SEQ ID NO:49 HCDR1 (IMGT) GFTFSSYA SEQ ID NO:50 HCDR2 (IMGT) ISGSGGST SEQ ID NO:77 HCDR3 (IMGT) ARREWWGESWLFDY SEQ ID NO:78 VH EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSS SEQ ID NO:79 DNA VH GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATG CCGACTCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGGGAGAAAGCTGGCTGTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:56 LCDR3 (Kabat) QQSYSTPLT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:59 LCDR3 (Chothia) SYSTPL SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:56 LCDR3 (IMGT) QQSYSTPLT SEQ ID NO:61 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:62 DNA VL GACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAGCTCCTGATCTACGCCGCTAGCTCGC TGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO:80 scFv (VH-linker-VL) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:81 DNA scFv GAAGTGCAGTTGCTGGAGTCAGGCGGAGGACTGGTGCAGCCCGGAGGATCGCTTCGCTTGAGCTGCGCAGCCTCAGGCTTTACCTTCTCCTCCTACGCCATGTCCTGGGTCAGACAGGCTCCCGGGAAGGGACTGGAATGGGTGTCCGCCATTAGCGGTTCCGGCGGAAGCACTTACTATGCCGAC TCTGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCCAAGAACACCCTGTATCTCCAAATGAATTCCCTGAGGGCCGAAGATACCGCGGTGTACTACTGCGCTAGACGGGAGTGGTGGGGAGAAAGCTGGCTGTTCGACTACTGGGGACAGGGCACTCTCGTGACTGTGTCCTCCGGTGGTGGT GGATCGGGGGGTGGTGGTTCGGGCGGAGGAGGATCTGGAGGAGGAGGGTCGGACATTCAAATGACTCAGTCCCCGTCCTCCCTCTCCGCCTCCGTGGGAGATCGCGTCACGATCACGTGCAGGCCAGCCAGAGCATCTCCAGCTACCTGAACTGGTACCAGCAGAAGCCAGGGAAGGCACCGAAG CTCCTGATCTACGCCGCTAGCTCGCTGCAGTCCGGCGTCCCCTTCACGGTTCTCGGGATCGGGCTCAGGCACCGACTTCACCCTGACCATTAGCAGCCTGCAGCCGGAGGACTTCGCGACATACTACTGTCAGCAGTCATACTCCACCCCTCTGACCTTCGGCCAAGGGACCAAAGTGGAGATCAAG duBCMA.4 SEQ ID NO:231 HCDR1 (Kabat) NHGMS SEQ ID NO:232 HCDR2 (Kabat) GIVYSGSTYYAASVKG SEQ ID NO:233 HCDR3 (Kabat) HGGESDV SEQ ID NO:234 HCDR1 (Chothia) GFALSNH SEQ ID NO:235 HCDR2 (Chothia) VYSGS SEQ ID NO:233 HCDR3 (Chothia) HGGESDV SEQ ID NO:236 HCDR1 (IMGT) GFALSNHG SEQ ID NO:237 HCDR2 (IMGT) IVYSGST SEQ ID NO:238 HCDR3 (IMGT) SAHGGESDV SEQ ID NO:239 VH EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS SEQ ID NO:262 DNA VH GAAGTGCAATTGGTGGAATCAGGGGGAGGACTTGTGCAGCCTGGAGGATCGCTGAGACTGTCATGTGCCGTGTCCGGCTTTGCCCTGTCCAACCACGGGATGTCCTGGGTCCGCCGCGCGCCTGGAAAGGGCCTCGAATGGGTGTCGGGTATTGTGTACAGCGGTAGCACCT ACTATGCCGCATCCGTGAAGGGGAGATTCACCATCAGCCGGGACAACTCCAGGAACACTCTGTACCTCCAATGAATTCGCTGAGGCCAGAGGACACTGCCATCTACTACTGCTCCGCGCATGGCGGAGAGTCCGACGTCTGGGGACAGGGGACCACGTGACCGTGTCTAGC SEQ ID NO:54 LCDR1 (Kabat) RASQSISSYLN SEQ ID NO:55 LCDR2 (Kabat) AASSLQS SEQ ID NO:240 LCDR3 (Kabat) QQSYSTPYT SEQ ID NO:57 LCDR1 (Chothia) SQSISSY SEQ ID NO:58 LCDR2 (Chothia) AAS SEQ ID NO:241 LCDR3 (Chothia) SYSTPY SEQ ID NO:60 LCDR1 (IMGT) QSISSY SEQ ID NO:58 LCDR2 (IMGT) AAS SEQ ID NO:240 LCDR3 (IMGT) QQSYSTPYT SEQ ID NO:242 VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK SEQ ID NO:263 DNA VL GACATCCAGCTCACCCAGTCCCCGAGCTCGCTGTCCGCCTCCGTGGGAGATCGGGTCACCATCACGTGCCGCGCCAGCCAGTCGATTTCCTCCTACCTGAACTGGTACCAACAGAAGCCCGGAAAAGCCCCGAAGCTTCTCATCTACGCCGCCTCGAGCC TGCAGTCAGGAGTGCCCTCACGGTTCTCCGGCTCCGGTTCCGGTACTGATTTCACCCTGACCATTTCCTCCCTGCAACCGGAGGACTTCGCTACTTACTACTGCCAGCAGTCGTACTCCACCCCCTACACTTTCGGACAAGGCACCAAGGTCGAAATCAAG SEQ ID NO:243 Linker ASGGGGSGGGGSGGGGS SEQ ID NO:200 scFv (VH-linker-VL) EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGG GGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK SEQ ID NO:201 DNA scFv Gaagtgcaattggtggaatcagggggaggacttgtgcagcctggaggatcgctgagactgtcatgtgccgtgtccggctttgccctgtccaaccacgggatgtcctgggtccgccgcgcgcctggaaagggcctcgaatgggtgtcgggtattgtgtacagcggtagcacctactatgc cgcatccgtgaaggggagattcaccatcagccgggacaactccaggaacactctgtacctccaaatgaattcgctgaggccagaggacactgccatctactactgctccgcgcatggcggagagtccgacgtctggggacaggggaccaccgtgaccgtgtctagcgcgtccggcggag gcggcagcgggggtggtggttcagggggcggcggatcggacatccagctcacccagtccccgagctcgctgtccgcctccgtggggagatcgggtcaccatcacgtgccgcgccagccagtcgatttcctcctacctgaactggtaccaacagaagcccggaaaagccccgaagcttctc atctacgccgcctcgagcctgcagtcaggagtgccctcacggttctccggctccggttccggtactgatttcaccctgaccatttcctccctgcaaccggaggacttcgctacttactactgccagcagtcgtactccaccccctacactttcggacaaggcaccaaggtcgaaatcaag duCD19.1 SEQ ID NO:244 HCDR1 GVSLPDYGVS SEQ ID NO:245 HCDR2 VIWGSETTYYQSSLKS SEQ ID NO:246 HCDR3 HYYYGGSYAMDY SEQ ID NO:295 HCDR1 (Kabat) DYGVS SEQ ID NO:245 HCDR2 (Kabat) VIWGSETTYYQSSLKS SEQ ID NO:246 HCDR3 (Kabat) HYYYGGSYAMDY SEQ ID NO:310 HCDR1 (Chothia) GVSLPDY SEQ ID NO:311 HCDR2 (Chothia) WGSET SEQ ID NO:246 HCDR3 (Chothia) HYYYGGSYAMDY SEQ ID NO:312 HCDR1 (IMGT) GVSLPDYG SEQ ID NO:313 HCDR2 (IMGT) IWGSETT SEQ ID NO:314 HCDR3 (IMGT) AKHYYYGGSYAMDY SEQ ID NO: 250 VH QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:315 DNA VH CAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGAGTGTCTCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACCAA TCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC SEQ ID NO:247 LCDR1 RASQDISKYLN SEQ ID NO:248 LCDR2 HTSRLHS SEQ ID NO:249 LCDR3 QQGNTLPYT SEQ ID NO:247 LCDR1 (Kabat) RASQDISKYLN SEQ ID NO:248 LCDR2 (Kabat) HTSRLHS SEQ ID NO:249 LCDR3 (Kabat) QQGNTLPYT SEQ ID NO:316 LCDR1 (Chothia) SQDISKY SEQ ID NO:317 LCDR2 (Chothia) HTS SEQ ID NO:318 LCDR3 (Chothia) GNTLPY SEQ ID NO:319 LCDR1 (IMGT) QDISKY SEQ ID NO:317 LCDR2 (IMGT) HTS SEQ ID NO:300 LCDR3 (IMGT) QQGNTLPYT SEQ ID NO:251 VL EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK SEQ ID NO:320 DNA VL GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGC TCCATTCTGGAATCCCTGCCAGGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAAA SEQ ID NO: 104 Linker GGGGSGGGGSGGGGS SEQ ID NO:211 scFv-domain of CAR2 - a. k. (linker underlined) EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGG SQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:305 scFv-domain CAR2 - n. c. GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTCTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCTCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCA GGTTCAGCGGTAGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTATTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAAAGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGA AGCCAGGTCCAACTCCAAGAAAGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGAGTGTCTCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGAGTGATTTGGGGCTCTGAGACTACTTACTACC AATCATCCCTCAAGTCACGCGTCACCATCTCAAAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGTGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGTACTCTGGTCACCGTGTCCAGC SEQ ID NO:225 Full CAR EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In some embodiments, disclosed herein is an isolated nucleic acid molecule comprising a first nucleic acid sequence encoding a first CAR polypeptide and a second nucleic acid sequence encoding a second CAR polypeptide, wherein the first CAR polypeptide comprises a first antigen-binding domain that is a BCMA binding domain (e.g., a human BCMA binding domain), a first transmembrane domain, and a first intracellular signaling domain, and wherein the second CAR polypeptide comprises a second antigen-binding domain that is a CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain. In some embodiments, the first CAR polypeptide comprises a VH comprising the HC CDR1, HC CDR2, and HC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, and a VL comprising the LC CDR1, LC CDR2, and LC CDR3 of a BCMA binding sequence set forth in Table 20 or 26, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243. In a certain embodiment, the first CAR polypeptide comprises a VH and VL comprising the amino acid sequences of SEQ ID NO: 239 and 242, respectively, wherein the VH and VL are connected by a linker comprising the amino acid sequence of SEQ ID NO: 243. In a certain embodiment, the first CAR polypeptide comprises an scFv comprising the amino acid sequence of SEQ ID NO: 200. In a certain embodiment, the first CAR polypeptide comprises the amino acid sequence of SEQ ID NO: 230 or 228. In some embodiments, the second CAR polypeptide comprises a CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and/or CDR3 LC of a CD19 binding sequence set forth in Table 19 or Table 22 (e.g., CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively). In some embodiments, the second CAR polypeptide comprises a VH and/or VL of the CD19 binding sequence set forth in Table 19 or Table 22 (e.g., a VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively), or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the second CAR polypeptide comprises a scFv of the CD19 binding sequence set forth in Table 19 or Table 22 (e.g., an scFv comprising the amino acid sequence of SEQ ID NO: 211), or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the second CAR polypeptide comprises a CAR polypeptide with a CD19 binding sequence shown in Table 19 or Table 22 (e.g., a CAR polypeptide comprising the amino acid sequence of SEQ ID NO: 225 or 229), or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 221 or 223. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 220 or 222 with or without the signal peptide of SEQ ID NO: 1.

Polyspecific CARs

In one embodiment, a CAR of the present invention is a multispecific CAR. In one embodiment, the multispecific CAR is a bispecific CAR. In one embodiment, the bispecific CAR comprises an antigen-binding domain that is a bispecific antibody molecule. The bispecific antibody is specific for up to two antigens. The bispecific antibody molecule is characterized by having a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as on different proteins (or on different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half-antibody that has binding specificity for a first epitope and a half-antibody that has binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half-antibody or fragment thereof that has binding specificity for a first epitope and a half-antibody or fragment thereof that has binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises an scFv or fragment thereof characterized by binding specificity to a first epitope and an scFv or fragment thereof characterized by binding specificity to a second epitope.

In some embodiments, a CAR of the present invention comprises an antigen-binding domain that is a multispecific (e.g., (bispecific or trispecific) antibodies. Protocols for producing bispecific or heterodimeric antibody molecules are known in the art, including, but not limited to, for example, the knob-into-hole approach as described, For example, in US 5731168; Fc pairing using directed electrostatic interaction as described, For example, in WO 09/089004, WO 06/106905 and WO 2010/129304; the formation of heterodimers by strand exchange domain engineering (SEED) as described, For example, in WO 07/110205; Fab fragment exchange as described, For example, in WO 08/119353, WO 2011/131746 and WO 2013/060867; dual antibody conjugates, For example, obtained by crosslinking antibodies to form a bispecific structure using a heterobifunctional reagent containing a reactive amino group and a reactive sulfhydryl group, as described, for example, in US 4,433,059; bispecific antibody determinants obtained by recombination of half-antibodies (heavy-light chain or Fab pairs) from different antibodies through a cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described, For example, in US 4444878; trifunctional antibodies, For example, three Fab' fragments cross-linked via reactive sulfhydryl groups as described, For example, in US5273743; biosynthetic binding proteins, For example, pairs of scFv cross-linked at the C-terminal tails, preferably by chemical cross-linking via disulfide bonds or reactive amino groups as described, For example, in US5534254; bifunctional antibodies, For example, Fab fragments with different binding specificities dimerized by leucine zippers (For example, c-fos and c-jun), replacing the constant domain as described, for example, in US5582996; bispecific and oligospecific mono- and oligovalent receptors, For example, VH-CH1 regions of two antibodies (two Fab fragments) linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with associated light chains as described, For example, in US5591828; bispecific DNA-antibody conjugates, For example, obtained by cross-linking antibodies or Fab fragments with a double-stranded DNA fragment as described, For example, in US5635602; bispecific fusion proteins, For example, an expression construct comprising two scFvs with a hydrophilic helical peptide linker between them and the entire constant region as described, For example, in US5637481; multivalent and multispecific binding proteins, For example, dimers of polypeptides having a first domain with a binding region of the variable region of an Ig heavy chain and a second domain with a binding region of the variable region of an Ig light chain, generally referred to as diabodies (higher order structures to create bispecific, trispecific or tetraspecific molecules are also encompassed, as described, for example, in US5837242; miniantibody constructs with linked VL and VH chains further linked by peptide spacers to the hinge region and CH3 region of the antibody, which can dimerize to form bispecific/multivalent molecules, as described, For example, in US5837821; VH and VL domains linked by a short peptide linker (For example, of 5 or 10 amino acids) or no linker at all, in any orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described, For example, in US5844094; a strand of VH domains (or VL domains in family members) linked by peptide bonds with crosslinkable groups at the C-terminus, further associated with VL domains to form sequentially linked Fv (or scFv), as described, For example, in US5864019; and single-chain binding polypeptides with both VH and VL domains linked by a peptide linker, combined into multivalent structures by non-covalent or chemical cross-linking to form, For example, homobivalent, heterobivalent, trivalent and tetravalent structures using both scFv-type and diabody-type formats as described, For example, in US5869620. Additional exemplary polyspecific and bispecific molecules and methods for making them can be found, for example, in US 5,910,573, US 5,932,448, US 5,959,083, US 5,989,830, US 6,005,079, US 6,239,259, US 6,294,353, US 6,333,396, US 6,476,198, US 6,511,663, US 6,670,453, US 6,743,896, US 6,809,185, US 6,833,441, US 7,129,330, US 7,183,076, US 7,521,056, US 7,527,787, US 7,534,866, US 7,612,181, US 2002,004,587 A1, US 2002076406 A1, US 2002103345 A1, US 2003207346 A1, US 2003211078 A1, US 2004219643 A1, US 2004220388 A1, US 2004242847 A1, US 2005003403 A1, US 2005004352 A1, US 2005069552 A1, US 2005079170 A1, US 2005100543 A1, US 2005136049 A1, US 2005136051 A1, US 2005163782 A1, US 2005266425 A1, US 2006083747 A1, US 2006120960 A1, US 2006204493 A1, US 2006263367 A1, US 2007004909 A1, US 2007087381 A1, US 2007128150 A1, US 2007141049 A1, US 2007154901 A1, US 2007274985 A1, US 2008050370 A1, US 2008069820 A1, US 2008152645 A1, US 2008171855 A1, US 2008241884 A1, US 2008254512 A1, US 2008260738 A1, US 2009130106 A1, US 2009148905 A1, US 2009155275 A1, US 2009162359 A1, US 2009162360 A1, US 2009175851 A1, US 2009175867 A1, US 2009232811 A1, US 2009234105 A1, US 2009263392 A1, US 2009274649 A1, EP 346087 A2, WO 0006605 A2, WO 02072635 A2, WO 04081051 A1, WO 06020258 A2, WO 2007044887 A2, WO 2007095338 A2, WO 2007137760 A2, WO 2008119353 A1, WO 2009021754 A2, WO 2009068630 A1, WO 9103493 A1, WO 9323537 A1, WO 9409131 A1, WO 9412625 A2, WO 9509917 A1, WO 9637621 A2, WO 9964460 A1. The contents of the above-mentioned applications are incorporated herein by reference in their entirety.

In each antibody or antibody fragment (e.g., scFv) in the bispecific antibody molecule, the VH may be located upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) has an organization such that its VH (VH ₁ ) is located upstream of its VL (VL ₁ ), and the downstream antibody or antibody fragment (e.g., scFv) has an organization such that its VL (VL ₂ ) is located upstream of its VH (VH ₂ ), such that the bispecific antibody molecule as a whole has an organization of VH ₁ -VL ₁ -VL ₂ -VH ₂ . In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) has an organization such that its VL (VL ₁ ) is located above its VH (VH ₁ ), and the downstream antibody or antibody fragment (e.g., scFv) has an organization such that its VH (VH ₂ ) is located above its VL (VL ₂ ), such that the bispecific antibody molecule as a whole has an organization of VL ₁ -VH ₁ -VH ₂ -VL ₂ . The linker is optionally located between two antibodies or antibody fragments (e.g., scFv), such as between VL ₁ and VL ₂ if the construct has an organization of VH ₁ -VL ₁ -VL ₂ -VH ₂ , or between VH ₁ and VH ₂ if the construct has an organization of VL ₁ -VH ₁ -VH ₂ -VL ₂ . The linker may be a linker as described herein, such as a (Gly ₄ -Ser)n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 26). Typically, the linker between two scFvs should be of sufficient length to prevent mispairing between the domains of the two scFvs. The linker is optionally located between the VL and VH of the first scFv. The linker is optionally located between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more linkers may be the same or different. Accordingly, in some embodiments, the bispecific CAR comprises a VL, a VH and optionally one or more linkers in an organization as described herein.

In one aspect, the bispecific antibody molecule is characterized by having a first immunoglobulin variable domain sequence, e.g., an scFv, that has binding specificity for BCMA, e.g., comprises an scFv described herein or comprises a light chain CDR and/or a heavy chain CDR of an scFv for BCMA described herein, and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on a different antigen. In one aspect, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on AML cells, e.g., an antigen other than BCMA. For example, the second immunoglobulin variable domain sequence has binding specificity for CD123. As another example, the second immunoglobulin variable domain sequence has binding specificity for CLL-1. As another example, the second immunoglobulin variable domain sequence is characterized by binding specificity to CD34. As another example, the second immunoglobulin variable domain sequence is characterized by binding specificity to FLT3. For example, the second immunoglobulin variable domain sequence is characterized by binding specificity to the folate receptor beta. In some aspects, the second immunoglobulin variable domain sequence is characterized by binding specificity to an antigen expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

CAR in diatel format

In some embodiments, a CAR of the present invention is a bispecific CAR. In some embodiments, a CAR of the present invention is a diabody format CAR. In some embodiments, a diabody format CAR comprises an antigen-binding domain that binds a first antigen and a second antigen. In some embodiments, the antigen-binding domain comprises VH1, VL1, VH2, and VL2, wherein VH1 and VL1 bind the first antigen and VH2 and VL2 bind the second antigen. In some embodiments, the antigen-binding domain has an organization of VH1-optionally Linker 1 ("L1")-VH2-optionally Linker 2 ("L2")-VL2-optionally Linker 3 ("L3")-VL1 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH1 - linker 1 ("L1") - VH2 - linker 2 ("L2") - VL2 - linker 3 ("L3") - VL1 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VH1-L1-VL2-L2-VH2-L3-VL1 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VL1-L1-VH2-L2-VL2-L3-VH1 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VL1-L1-VL2-L2-VH2-L3-VH1 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VH2-L1-VH1-L2-VL1-L3-VL2 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VH2-L1-VL1-L2-VH1-L3-VL2 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VL2-L1-VH1-L2-VL1-L3-VH2 organization from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has a VL2-L1-VL1-L2-VH1-L3-VH2 organization from the N-terminus to the C-terminus. In some embodiments, the variable regions are fused by a linker comprising the amino acid sequence GGGGSGGGGS (SEQ ID NO: 5). In some embodiments, the variable regions are fused by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 63). In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, L2 comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, L3 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, VH1, VL1, VH2, or VL2 comprises a CDR, VH, or VL sequence disclosed herein or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, a diabody disclosed herein comprises an engineered disulfide bridge, such as to stabilize the diabody and/or to promote proper pairing of the VH and VL. In some embodiments, the engineered disulfide bridge is between the variable region that is located most proximal to the hinge region (e.g., the VH or VL region that is located most proximal to the hinge region) and its corresponding pairing partner (e.g., the corresponding VL or the corresponding VH).

In some embodiments, the first antigen and the second antigen are different. In some embodiments, the first or second antigen is selected from an antigen expressed on B cells, an antigen expressed on acute myeloid leukemia cells, or an antigen on solid tumor cells. In some embodiments, the first or second antigen is selected from CD10, CD19, CD20, CD22, CD34, CD123, BCMA, FLT-3, ROR1, CD79b, CD179b, CD79a, CD34, CLL-1, folate receptor beta, FLT3, EGFRvIII, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptides, sTn-O-glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y antigen, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB (eg, ERBB2), Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, human chorionic gonadotropin beta, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mutant hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC.

In some embodiments, the first antigen is BCMA and the second antigen is CD19. In some embodiments, the CAR comprises an antigen-binding domain that binds to BCMA and CD19. In some embodiments, the antigen-binding domain comprises VH ₁ and VL ₁ that bind to BCMA ("VH for BCMA" and "VL for BCMA") and VH ₂ and VL ₂ that bind to CD19 ("VH for CD19" and "VL for CD19"). In some embodiments, the antigen-binding domain has an organization of VH for BCMA-optionally Linker 1 ("L1")-VH for CD19-optionally Linker 2 ("L2")-VL for CD19-optionally Linker 3 ("L3")-VL for BCMA from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for BCMA - optionally L1 - VL for CD19 - optionally L2 - VH for CD19 - optionally L3 - VL for BCMA from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for BCMA - optionally L1 - VH for CD19 - optionally L2 - VL for CD19 - optionally L3 - VH for BCMA from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for BCMA - optionally L1 - VL for CD19 - optionally L2 - VH for CD19 - optionally L3 - VH for BCMA from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for CD19 - optionally L1 - VH for BCMA - optionally L2 - VL for BCMA - optionally L3 - VL for CD19 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for CD19 - optionally L1 - VL for BCMA - optionally L2 - VH for BCMA - optionally L3 - VL for CD19 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for CD19 - optionally L1 - VH for BCMA - optionally L2 - VL for BCMA - optionally L3 - VH for CD19 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for CD19 - optionally L1 - VL for BCMA - optionally L2 - VH for BCMA - optionally L3 - VH for CD19 from N-terminus to C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for BCMA - Linker 1 ("L1") - VH for CD19 - Linker 2 ("L2") - VL for CD19 - Linker 3 ("L3") - VL for BCMA from N-terminus to C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for BCMA - L1 - VL for CD19 - L2 - VH for CD19 - L3 - VL for BCMA from N-terminus to C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for BCMA - L1 - VH for CD19 - L2 - VL for CD19 - L3 - VH for BCMA from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for BCMA - L1 - VL for CD19 - L2 - VH for CD19 - L3 - VH for BCMA from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for CD19 - L1 - VH for BCMA - L2 - VL for BCMA - L3 - VL for CD19 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VH for CD19 - L1 - VL for BCMA - L2 - VH for BCMA - L3 - VL for CD19 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for CD19-L1-VH for BCMA-L2-VL for BCMA-L3-VH for CD19 from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain has an organization of VL for CD19-L1-VL for BCMA-L2-VH for BCMA-L3-VH for CD19 from the N-terminus to the C-terminus. In some embodiments, the variable regions are fused by a linker comprising the amino acid sequence of SEQ ID NO: 5 or 63 or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, L2 comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, L3 comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the VH for BCMA comprises the CDR or VH sequences disclosed herein, such as the CDR or VH sequences disclosed in Tables 3-15, 19, 20, 22, 26, and 31, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the VL for BCMA comprises the CDR or VL sequences disclosed herein, such as the CDR or VL sequences disclosed in Tables 3-15, 19, 20, 22, 26, and 31, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the VH for CD19 comprises the CDR or VH sequences disclosed herein, such as the CDR or VH sequences disclosed in Tables 2, 19, 22, and 31, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the VL for CD19 comprises the CDR or VL sequences disclosed herein, such as the CDR or VL sequences disclosed in Tables 2, 19, 22, and 31, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, a CAR, e.g., a diabody format CAR, further comprises a hinge region, a transmembrane domain, and/or an intracellular signaling domain. In some embodiments, the hinge region comprises a CD8 hinge region. In some embodiments, the hinge region comprises a hinge region sequence disclosed herein, e.g., the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain sequence disclosed herein, e.g., the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the intracellular signaling domain comprises the 4-1BB intracellular domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain sequence disclosed herein, such as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the intracellular signaling domain comprises the CD3 intracellular domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain sequence disclosed herein, such as the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto.

Exemplary diabody sequences are disclosed in Table 31. In some embodiments, a CAR comprises an antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 321-330, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 339-348, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity thereto.

Table 31. Illustrative CAR components in diabody format SEQ ID NO Description Amino acid sequence* SEQ ID NO:321 Antigen binding domain of JL1 QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGG GGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVLGGGGSGGGG SGGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDV WGQGTLVTVSSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIK SEQ ID NO:339 Full-size CAR JL1 in diatel format QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAAD TAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDV SNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGY ALHDDYYGLDVWGQGTLVTVSSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:322 Antigen binding domain of JL2 EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKGGGGSQVQLQESGGG VVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGGSGGGGS GGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVLGG GGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:340 Full-size CAR JL2 in diatel format EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVF GSGTKVTVLGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:323 Antigen binding domain of JL3 QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGG GGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLY VFGSGTKVTVLGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIK SEQ ID NO:341 Full-size CAR JL3 in diatel format QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAAD TAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISY DGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYY CSSYTSSSTLYVFGSGTKVTVLGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:324 Antigen binding domain of JL4 EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKGGGGSQSALTQPASV SGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVLGGGGSGGGGSGGGGSGGGGSQV QLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGG GGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:342 Full-size CAR JL4 in diatel format EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSG SKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWG QGTLVTVSSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:325 Antigen binding domain of JL5 QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVS SGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGS GGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQG TLVTVSSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVL SEQ ID NO:343 Full-size CAR JL5 in diatel format QVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYG GSYAMDYWGQGTLVTVSSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:326 Antigen binding domain of JL6 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVLGGGGSQVQLQE SGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGG SGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGS QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO:344 Full-size CAR JL6 in diatel format QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVLGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSS LKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQG TKLEIKGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:327 Antigen binding domain of JL7 QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVS SGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ GTKLEIKGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVL SEQ ID NO:345 Full-size CAR JL7 in diatel format QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV IWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQ QGNTLPYTFGQGTKLEIKGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:328 Antigen binding domain of JL8 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVLGGGGSEIVMTQ SPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQV QLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGS QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO:346 Full-size CAR JL8 in diatel format QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVLGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARF SSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTV SGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGT LVTVSSGGGGSQVQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:329 Antigen binding domain of JL9 QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSG GGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGGGGSGGGGS GGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYW GQGTLVTVSSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIK SEQ ID NO:347 Full-size CAR JL9 in diatel format QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAA DTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWW GESWLFDYWGQGTLVTVSSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:330 Antigen binding domain of JL10 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSS GGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGG SGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYW GQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGCGTKVEIK SEQ ID NO:348 Full-size CAR JL10 in diatel format EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHT SRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYY GGSYAMDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGCGTKVEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 250 VH for CD19 (CTL119) QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:251 VL for CD19 (CTL119) EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK SEQ ID NO:331 VH variant for CD19 (CTL119) QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKCLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS SEQ ID NO:332 VL variant for CD19 (CTL119) EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGCGTKLEIK SEQ ID NO:93 VH for BCMA (PI61) QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO: 102 VL for BCMA (PI61) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGSGTKVTVL SEQ ID NO:333 VH variant for BCMA (PI61) QVQLQESGGGVVQPGRSLRLSCAASGFFSSYGMHWVRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGGSGYALHDDYYGLDVWGQGTLVTVSS SEQ ID NO:334 Option VL for BCMA (PI61) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLYVFGCGTKVTVL SEQ ID NO:78 VH for BCMA (R1G5) EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSS SEQ ID NO:61 VL for BCMA (R1G5) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK SEQ ID NO:335 VH variant for BCMA (R1G5) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARREWWGESWLFDYWGQGTLVTVSS SEQ ID NO:336 Option VL for BCMA (R1G5) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGCGTKVEIK SEQ ID NO:5 Linker GGGGSGGGGS SEQ ID NO:63 Linker GGGGSGGGGSGGGGSGGGGS *: VH sequences are underlined and VL sequences are double underlined. CD19 binding sequences (VH and VL) are shown in italics.

Chimeric TCR

In one aspect, the antibodies and antibody fragments of the present invention can be grafted onto one or more constant domains of a T cell receptor ("TCR") chain, such as a TCR alpha chain or a TCR beta chain, to create a chimeric TCR. Without being limited by theory, it is believed that chimeric TCRs will signal through the TCR complex upon antigen binding. For example, an scFv disclosed herein can be grafted onto a constant domain, such as at least a portion of an extracellular constant domain, a transmembrane domain, and a cytoplasmic domain, of a TCR chain, such as a TCR alpha chain and/or a TCR beta chain. As another example, an antibody fragment, such as the VL domain described herein, can be grafted onto the constant domain of a TCR alpha chain, and an antibody fragment, such as the VH domain described herein, can be grafted onto the constant domain of a TCR beta chain (or, alternatively, the VL domain can be grafted onto the constant domain of a TCR beta chain and the VH domain can be grafted onto the alpha chain of a TCR). As another example, a CDR of an antibody or antibody fragment, such as a CDR of an antibody or antibody fragment described herein, can be grafted onto the alpha and/or beta chain of a TCR to create a chimeric TCR. For example, the LCDRs disclosed herein can be grafted onto the variable domain of a TCR alpha chain, and the HCDRs disclosed herein can be grafted onto the variable domain of a TCR beta chain, or vice versa. Such chimeric TCRs can be generated by methods known in the art (e.g., Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; Aggen et al, Gene Ther. 2012 Apr; 19(4):365-74).

Additional embodiments

In one aspect, a cell expressing a CAR as described herein may further comprise a second CAR, such as a second CAR that comprises a different antigen binding domain, such as for the same target (BCMA) or a different target (e.g., CD19, CD20, or CS-1 or other multiple myeloma targets, such as kappa light chain, CD138, Lewis Y antigen, or CD38 (Garfall et al., Discovery Medicine, 2014, 17(91):37-46)). In one embodiment, a cell expressing a CAR comprises a first CAR that targets a first antigen and comprises an intracellular signaling domain that has a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain that has a primary signaling domain but not a costimulatory signaling domain. Without being limited by theory, the placement of a costimulatory signaling domain, such as 4-1BB, CD28, CD27, or OX-40, in a first CAR and a primary signaling domain, such as CD3 zeta, in a second CAR can limit the activity of the CAR to cells that express both targets. In one embodiment, a cell expressing a CAR comprises a first CAR for BCMA that comprises a BCMA binding domain, a transmembrane domain, and a costimulatory domain, and a second CAR that targets an antigen other than BCMA (e.g., an antigen expressed on leukemia or lymphoma cells, such as CD19, CD20, CS-1, kappa light chain, CD139, Lewis antigen Y, or CD38) and comprises an antigen binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, a cell expressing a CAR comprises a first CAR for BCMA that comprises a BCMA binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets an antigen other than BCMA (e.g., an antigen expressed on leukemia or lymphoma cells, such as CD19, CD20, CS-1, kappa light chain, CD139, Lewis antigen Y, or CD38) and comprises an antigen binding domain for the antigen, a transmembrane domain, and a costimulatory signaling domain. In one embodiment, a cell expressing a CAR comprises a CAR for BCMA described herein and a CAR that targets CD19 (a CD19 CAR).

In one embodiment, a cell expressing a CAR comprises a CAR for BCMA described herein and an inhibitory CAR. In one embodiment, the inhibitory CAR comprises an antigen-binding domain that binds an antigen found on normal cells but not on cancer cells. In one embodiment, the inhibitory CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of an inhibitory CAR can be the intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecules, MHC class II molecules, GAL9, adenosine, and TGFR-beta.

In one embodiment, if a cell expressing a CAR comprises two or more different CARs, the antigen binding domains of the different CARs may be such that the antigen binding domains do not interact with each other. For example, a cell expressing a first and a second CAR may have an antigen binding domain of the first CAR, for example, as a fragment, for example, an scFv, that does not associate with the antigen binding domain of the second CAR, for example, the antigen binding domain of the second CAR is VHH.

In some embodiments, the antigen-binding domain comprises single-domain antigen-binding (SDAB) molecules, including molecules in which the complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, variable domains of heavy chains, binding molecules naturally lacking light chains, individual domains derived from traditional 4-chain antibodies, engineered domains, and single-domain frameworks other than those derived from antibodies. SDAB molecules may be any molecule known in the art or any single-domain molecule that is later known. SDAB molecules may be derived from any species, including, but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. The term also encompasses naturally occurring single-domain antibody molecules from species other than Camelidae and sharks.

In one aspect, the SDAB molecule can be derived from a variable region of an immunoglobulin found in fish, such as that derived from an immunoglobulin isotype known as a novel antigen receptor (NAR) found in shark serum. Methods for producing single-domain molecules derived from the variable region of a NAR ("IgNAR") are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, the SDAB molecule is a naturally occurring single-domain antigen-binding molecule known as a light chain-less heavy chain. Such single-domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For the sake of clarity, this variable domain obtained from a light chain-less heavy chain molecule is referred to herein as a VHH or nanobody to distinguish it from the traditional four-chain immunoglobulin VH. Such a VHH molecule may be obtained from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other species besides Camelidae may produce light chain-less heavy chain molecules; such VHHs are within the scope of the present invention.

SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, deimmunized, and/or generated in vitro (e.g., selected by phage display).

It has also been found that in cells having a plurality of membrane-embedded chimeric receptors comprising an antigen-binding domain, interactions between the antigen-binding domains of the receptors may be undesirable, for example, because they inhibit the ability of one or more antigen-binding domains to bind their cognate antigens. Accordingly, cells having first and second non-naturally occurring membrane-embedded chimeric receptors comprising antigen-binding domains for which such interactions are minimized are disclosed herein. Nucleic acids encoding first and second non-naturally occurring membrane-embedded chimeric receptors comprising antigen-binding domains for which such interactions are minimized are also disclosed herein, as well as methods for producing and using such cells and nucleic acids. In one embodiment, the antigen binding domain of one of said first and said second non-naturally occurring membrane-integrated chimeric receptors comprises an scFv and the other comprises a single VH domain, such as a single VH domain of camelid, shark or lamprey or a single VH domain derived from a human or mouse sequence.

In some embodiments, the claimed invention provides a first and a second CAR, wherein the antigen-binding domain of one of said first CAR and said second CAR does not comprise a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR is an scFv, and the other is not an scFv. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises a single VH domain, such as a single VH domain of camelids, sharks or lampreys, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises a nanobody. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises a camelid VHH domain.

In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises an scFv and the other comprises a single VH domain, such as a single VH domain of camelids, sharks or lampreys or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises an scFv and the other comprises a nanobody. In some embodiments, the antigen-binding domain of one of said first CAR and said second CAR comprises an scFv and the other comprises a camelid VHH domain.

In some embodiments, the binding of the antigen-binding domain of said first CAR, when present on the surface of a cell, to its cognate antigen is not substantially reduced by the presence of said second CAR. In some embodiments, the binding of the antigen-binding domain of said first CAR to its cognate antigen in the presence of said second CAR is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen-binding domain of said first CAR to its cognate antigen in the absence of said second CAR.

In some embodiments, the antigen-binding domains of said first CAR and said second CAR, when present on the surface of a cell, associate with each other to a lesser extent than if they were both antigen-binding domains of an scFv. In some embodiments, the antigen-binding domains of said first CAR and said second CAR associate with each other to a lesser extent than if they were both antigen-binding domains of an scFv.

In another aspect, a CAR-expressing cell described herein may further express another agent, such as an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule, such as an agent described herein. Inhibitory molecules, such as PD1, in some embodiments may reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecule, MHC class II molecule, GAL9, adenosine, and TGFR-beta. In one embodiment, an agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to a cell, e.g., an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., from an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, an MHC class I molecule, an MHC class II molecule, GAL9, adenosine, and TGFR-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain as described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, ICOS, or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide that is an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain described herein). In some embodiments, a cell expressing a CAR described herein comprises a switch co-stimulatory receptor, e.g., as described in WO 2013/019615, which is incorporated herein by reference in its entirety. PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765–75). Two PD1 ligands, PD-L1 and PD-L2, have been shown to suppress T cell activation when bound to PD1 (Freeman et al. 2000 J Exp Med 192:1027–34; Latchman et al. 2001 Nat Immunol 2:261–8; Carter et al. 2002 Eur J Immunol 32:634–43). PD-L1 is highly expressed in a variety of human cancers (Dong et al. 2003 J Mol Med 81:281–7; Blank et al. 2005 Cancer Immunol. Immunother 54:307–314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be reversed by inhibiting local interaction of PD1 with PD-L1.

In one embodiment, an agent comprising an extracellular domain (ECD) of an inhibitory molecule, such as programmed cell death protein 1 (PD1), can be fused to a transmembrane domain and intracellular signaling domains, such as 4-1BB and CD3-zeta (also referred to herein as a PD1-based CAR). In one embodiment, the PD1-based CAR, when used in combinations with the BCMA CAR described herein, improves the persistence of a cell expressing the CAR, such as a T cell or NK cell. In one embodiment, the CAR is a PD1-based CAR comprising the extracellular domain of PD1 indicated by underlining in SEQ ID NO: 24. In one embodiment, the PD1-based CAR comprises the amino acid sequence of SEQ ID NO: 24.

In one embodiment, a PD1-based CAR comprises the amino acid sequence shown below (SEQ ID NO: 22).

In one embodiment, the agent comprises a nucleic acid sequence encoding a PD1-based CAR, such as a PD1-based CAR described herein. In one embodiment, the nucleic acid sequence for a PD1-based CAR is set forth as SEQ ID NO: 23, with the PD1 ECD underlined.

In another aspect, the present invention provides a population of cells expressing a CAR, such as CAR-T cells or NK cells expressing a CAR. In some embodiments, the population of cells expressing a CAR comprises a mixture of cells expressing different CARs. For example, in one embodiment, a population of cells expressing a CAR (e.g., CAR-T cells or NK cells expressing a CAR) can comprise a first cell expressing a CAR having a BCMA binding domain described herein and a second cell expressing a CAR having a different BCMA binding domain, such as a BCMA binding domain described herein that is different from the BCMA binding domain of the CAR expressed by the first cell. As another example, a population of cells expressing a CAR can comprise a first cell expressing a CAR that comprises a BCMA binding domain, such as those described herein, and a second cell expressing a CAR that comprises an antigen-binding domain for a target other than BCMA (e.g., CD19, CD20, CS-1, kappa light chain, CD139, Lewis antigen Y, or CD38). In one embodiment, the population of cells expressing a CAR comprises a first cell expressing a CAR that comprises a BCMA binding domain, such as those described herein, and a second cell expressing a CAR that comprises an antigen-binding domain that targets CD19 (a CD19 CAR). In one embodiment, the population of cells expressing a CAR comprises, for example, a first cell expressing a CAR that comprises a primary intracellular signaling domain and a second cell expressing a CAR that comprises a secondary signaling domain.

In another aspect, the present invention provides a population of cells, wherein at least one cell in the population expresses a CAR having a BCMA binding domain as described herein, and a second cell expresses another agent, such as an agent that enhances the activity of a cell expressing the CAR. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, for example, may, in some embodiments, reduce the ability of a cell expressing a CAR to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecule, MHC class II molecule, GAL9, adenosine, and TGFR-beta. In one embodiment, an agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to a cell, e.g., an intracellular signaling domain as described herein. In one embodiment, the agent comprises a first polypeptide, e.g., from an inhibitory molecule such as PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, an MHC class I molecule, an MHC class II molecule, GAL9, adenosine, and TGFR-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain as described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, ICOS or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide that is an intracellular signaling domain described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain described herein).

In one aspect, the present invention provides methods comprising administering a population of cells expressing a CAR (e.g., CAR T cells or NK cells expressing a CAR), e.g., a mixture of cells expressing different CARs, in combination with another agent, e.g., a kinase inhibitor, such as a kinase inhibitor described herein. In another aspect, the present invention provides methods comprising administering a population of cells, wherein at least one cell in the population expresses a CAR having an antigen-binding domain for a cancer-associated antigen described herein, and a second cell expressing another agent, e.g., an agent that enhances the activity of a cell expressing the CAR, in combination with the other agent, e.g., a kinase inhibitor, such as a kinase inhibitor described herein.

CARs containing natural killer cell receptors (NKRs)

In one embodiment, a CAR molecule described herein comprises one or more natural killer cell receptor (NKR) components, thereby forming an NKR CAR. The NKR component can be a transmembrane domain, a hinge domain, or a cytoplasmic domain of any of the following natural killer cell receptors: an immunoglobulin-like killer cell receptor (KIR), e.g., KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; a natural cytotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46; the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; the Fc receptor (FcR), such as CD16 and CD64; and the Ly49 receptors, such as LY49A, LY49C. The NKR CAR molecules described herein can interact with an adaptor molecule or an intracellular signaling domain, such as DAP12. Exemplary configurations and sequences of CAR molecules comprising NKR components are described in International Publication No. WO2014/145252, the contents of which are incorporated herein by reference.

Scaffold structures other than antibodies

In some embodiments, the antigen-binding domain comprises a framework other than an antibody, such as fibronectin, ankyrin, a domain antibody, lipocalin, a small modular immunopharmaceutical, a maxibody, protein A, or an affilin. The framework other than an antibody has the ability to bind to a target antigen on a cell. In some embodiments, the antigen-binding domain is a polypeptide or fragment thereof from a naturally occurring protein expressed on a cell. In some embodiments, the antigen-binding domain comprises a framework other than an antibody. A wide variety of frameworks other than antibodies can be used, so long as the resulting polypeptide contains at least one binding region that specifically binds to a target antigen on a target cell.

Non-antibody scaffolds include fibronectin (Novartis, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA and Ablynx NV, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxi antibodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

Strategies for regulation of chimeric antigen receptors

There are multiple ways in which CAR activity patterns can be regulated. In some embodiments, a regulated CAR (RCAR), in which CAR activity can be controlled, is desirable to optimize the safety and efficacy of CAR therapy. For example, induction of apoptosis using, for example, a caspase fused to a dimerization domain (see, for example, Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the present invention. In another example, cells expressing a CAR may also express an inducible caspase-9 (iCaspase-9) molecule that, upon administration of a dimerizing drug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)) results in caspase-9 activation and cell apoptosis. The iCaspase-9 molecule contains a chemical inducer of dimerization (CID) binding domain that mediates dimerization in the presence of CID. This results in inducible and selective depletion of CAR-expressing cells. In some cases, the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the vector(s) encoding the CAR. In some cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the vector encoding the CAR. iCaspase-9 may provide a protective switch to avoid potential toxicity in CAR-expressing cells. See, e.g., Song et al. Cancer Gene Ther. 2008; 15(10):667–75; Clinical Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365:1673–83.

Alternative strategies for regulating the CAR therapy of the present invention include the use of small molecules or antibodies that deactivate or turn off CAR activity, such as by removing cells expressing the CAR, such as by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). For example, cells expressing the CAR described herein may also express an antigen that is recognized by molecules capable of inducing cell death, such as ADCC or complement-induced cell death. For example, cells expressing the CAR described herein may also express a receptor that can be targeted by an antibody or antibody fragment. Examples of such receptors include EpCAM, VEGFR, integrins (e.g., ανβ3, α4, αΙ¾β3, α4β7, α5β1, ανβ3, αν integrins), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD1 1, CD1 1 a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/receptor IgE, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7 and EGFR and truncated variants thereof (e.g., variants that retain one or more extracellular epitopes but lack one or more regions in the cytoplasmic domain). For example, the CAR-expressing cells described herein may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capacity but retains an epitope recognized by molecules capable of inducing ADCC, such as cetuximab (ERBITUX®), such that administration of cetuximab induces ADCC and subsequent depletion of the CAR-expressing cell population (see, e.g., WO2011/056894 and Jonnalagadda et al., Gene Ther. 2013; 20(8)853-860). Another strategy involves expressing a highly compact marker/"suicide" gene that combines target epitopes of both the CD32 and CD20 antigens in cells expressing the CARs described herein that bind to rituximab, resulting in selective depletion of the CAR expressing cell population, such as by ADCC (see, e.g., Philip et al., Blood. 2014; 124(8)1277-1287). Other methods of depleting the CAR expressing cell population described herein include administering CAMPATH®, an anti-CD52 monoclonal antibody that selectively binds to mature lymphocytes, such as CAR expressing cells, and targets them for destruction, such as by inducing ADCC. In other embodiments, CAR expressing cells can be selectively targeted with a CAR ligand, such as an anti-idiotype antibody. In some embodiments, an anti-idiotypic antibody may mediate effector cell activity, such as ADCC or ADC activity, thereby reducing the number of CAR-expressing cells. In other embodiments, a CAR ligand, such as an anti-idiotypic antibody, may be linked to an agent that induces cell killing, such as a toxin, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecules themselves may be configured such that activity can be adjusted, such as by being turned on or off, as described below.

In some embodiments, an RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, such as an antigen-binding domain and an intracellular signaling domain, are separated into separate polypeptides or components. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can link the polypeptides to each other, such as can link the antigen-binding domain to the intracellular signaling domain. Further description and exemplary configurations of such regulable CARs are provided herein and in International Publication No. WO 2015/090229, incorporated herein by reference in its entirety.

In one embodiment, the RCAR comprises two polypeptides or components: 1) an intracellular signaling component comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain as described herein, and a first switch domain; 2) an antigen-binding component comprising an antigen-binding domain, e.g., that targets a tumor antigen as described herein, and a second switch domain. The RCAR optionally comprises a transmembrane domain as described herein. In one embodiment, the transmembrane domain can be located in the intracellular signaling component, the antigen-binding component, or both. (Unless otherwise indicated, when describing components or elements of an RCAR herein, the order may be as provided, but other orders are also included. In other words, in one embodiment, the order is as set forth in the text, but in other embodiments, the order may be different. For example, the order of elements on one side of the transmembrane region may differ from the example, for example, the placement of the switch domain relative to the intracellular signaling domain may be different, such as reversed.)

In one embodiment, the first and second switch domains may form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be a homodimerization switch, such as if the first and second switch domains are the same, or a heterodimerization switch, such as if the first and second switch domains are different from each other.

In some embodiments, an RCAR may comprise a "multi-switch." The multi-switch may comprise heterodimerization switch domains or homodimerization switch domains. The multi-switch comprises a plurality, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, switch domains separately in a first component, such as an antigen-binding component, and a second component, such as an intracellular signaling component. In one embodiment, the first component may comprise a plurality of first switch domains, such as FKBP-based switch domains, and the second component may comprise a plurality of second switch domains, such as FRB-based switch domains. In one embodiment, the first component may comprise a first and a second switch domain, such as an FKBP-based switch domain and an FRB-based switch domain, and the second component may comprise a first and a second switch domain, such as an FKBP-based switch domain and an FRB-based switch domain.

In one embodiment, the intracellular signaling component comprises one or more intracellular signaling domains, such as a primary intracellular signaling domain and one or more costimulatory signaling domains.

In one embodiment, the antigen-binding component may comprise one or more intracellular signaling domains, such as one or more costimulatory signaling domains. In one embodiment, the antigen-binding component comprises a plurality, such as 2 or 3, costimulatory signaling domains described herein, such as selected from 4-1BB, CD28, CD27, ICOS, and OX40, and in some embodiments, the primary intracellular signaling domain is absent. In one embodiment, the antigen-binding component comprises the following costimulatory signaling domains in the extracellular to intracellular direction: 4-1BB-CD27; 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; CD28-4-1BB, or 4-1BB-CD28. In such embodiments, the intracellular binding component comprises a CD3 zeta domain. In one such embodiment, the RCAR comprises (1) an antigen binding component comprising an antigen binding domain, a transmembrane domain, and two costimulatory domains, as well as a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane or membrane anchoring domain and at least one primary intracellular signaling domain and a second switch domain.

In one embodiment, RCARs are provided wherein the antigen-binding component is not attached to the surface of the CAR cell. This allows a cell having an intracellular signaling component to successfully bind to one or more antigen-binding domains without transforming the cell with a sequence that encodes the antigen-binding component. In such embodiments, the RCAR comprises 1) an intracellular signaling component comprising a first switch domain, a transmembrane domain, an intracellular signaling domain, e.g., a primary intracellular signaling domain, and a first switch domain; and 2) an antigen-binding component comprising an antigen-binding domain and a second switch domain, wherein the antigen-binding component does not comprise a transmembrane or membrane anchor domain and optionally does not comprise an intracellular signaling domain. In some embodiments, the RCAR may further comprise 3) a second antigen-binding component comprising a second antigen-binding domain, e.g., a second antigen-binding domain that binds an antigen different from that bound by the antigen-binding domain; and a second switch domain.

Also provided herein are RCARs wherein the antigen-binding component has bispecific activation and targeting capability. In such an embodiment, the antigen-binding component may comprise a plurality, such as 2, 3, 4, or 5, antigen-binding domains, such as an scFv, wherein each antigen-binding domain binds to a target antigen, such as different antigens or the same antigen, such as the same or different epitopes on the same antigen. In one embodiment, the plurality of antigen-binding domains are in tandem, and a linker or hinge region is optionally located between every two consecutive antigen-binding domains. Suitable linkers and hinge regions are described herein.

In one embodiment, RCARs are provided that have a configuration that provides a proliferation switch. In such an embodiment, the RCAR comprises 1) an intracellular signaling component optionally comprising a transmembrane domain or a membrane anchor domain; one or more costimulatory signaling domains, such as those selected from 4-1BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen-binding component comprising an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain, such as a CD3-zeta domain, wherein the antigen-binding component does not comprise a switch domain or does not comprise a switch domain that dimerizes with a switch domain in the intracellular signaling component. In one embodiment, the antigen-binding component does not comprise a costimulatory signaling domain. In one embodiment, the intracellular signaling component comprises a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling component comprises a first switch domain of a heterodimerization switch, and the RCAR comprises a second intracellular signaling component that comprises a second switch domain of the heterodimerization switch. In such embodiments, the second intracellular signaling component comprises the same intracellular signaling domains as the intracellular signaling component. In one embodiment, the dimerization switch is intracellular. In one embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described herein, the first and second switching domains comprise an FKBP-FRB based switch as described herein.

Also provided herein are cells comprising the RCAR described herein. Any cell engineered to express RCAR can be used as an RCARX cell. In one embodiment, the RCARX cell is a T cell and is referred to as an RCAR-T cell. In one embodiment, the RCARX cell is an NK cell and is referred to as an RCAR-N cell.

Also provided herein are nucleic acids and vectors comprising sequences encoding an RCAR. Sequences encoding different components of an RCAR may be located on the same nucleic acid molecule, such as the same plasmid or vector, such as a viral vector, such as a lentiviral vector. In one embodiment, (i) a sequence encoding an antigen-binding component and (ii) a sequence encoding an intracellular signaling component may be located on the same nucleic acid, such as a vector. Production of the respective proteins may be achieved, such as by using separate promoters or by using a bicistronic transcription product (which may result in the production of two proteins by cleavage of a single translation product or by translation of two separate protein products). In one embodiment, a sequence encoding a cleavable peptide, such as a P2A or F2A sequence, is located between (i) and (ii). In one embodiment, the sequence encoding an IRES, such as an EMCV or EV71 IRES, is located between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, the first promoter is operably linked to (i) and the second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mRNAs.

Alternatively, the sequences encoding the different components of the RCAR may be located in different nucleic acid molecules, such as in different plasmids or vectors, such as in viral vectors, such as in lentiviral vectors. For example, (i) the sequence encoding the antigen-binding component may be located in a first nucleic acid, such as in a first vector, and (ii) the sequence encoding the intracellular signaling component may be located in a second nucleic acid, such as in a second vector.

Dimerization switches

Dimerization switches can be noncovalent or covalent. In a noncovalent dimerization switch, the dimerizing molecule facilitates a noncovalent interaction between the switching domains. In a covalent dimerization switch, the dimerizing molecule facilitates a covalent interaction between the switching domains.

In one embodiment, the RCAR comprises a FKBP/FRAP or FKBP/FRB-based dimerization switch. FKBP12 (FKBP or FK506-binding protein) is a widely distributed cytoplasmic protein that serves as the initial intracellular target of rapamycin, a natural product immunosuppressive drug. Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR). FRB is the 93-amino acid portion of FRAP that is sufficient to bind the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue . Proc Natl Acad Sci USA 92: 4947–51).

In some embodiments, a dimerizing molecule, such as rapamycin or a rapamycin analog, may be used for a FKBP/FRAP-based switch, such as a FKBP/FRB-based switch.

An illustrative amino acid sequence of FKBP is as follows:

D V P D Y A S L G G P S S P K K K R K V S R G V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S Y (SEQ ID NO: 275)

In some embodiments, the FKBP-based switch domain may comprise a fragment of FKBP that has the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or rapalog. In one embodiment, the FKBP-based switch domain comprises the following amino acid sequence:

V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S (SEQ ID NO: 276)

The amino acid sequence of FRB is as follows:

ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK (SEQ ID NO: 277)

As used herein, the term "FKBP/FRAP-based switch, e.g. FKBP/FRB-based" refers to a dimerization switch comprising a first switch domain that comprises a fragment of FKBP or an analog thereof that is capable of binding to FRB or a fragment or analog thereof in the presence of rapamycin or a rapalog, e.g. RAD001, and is characterized by at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the FKBP sequence of SEQ ID NO: 275 or 276 or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue therefrom; and a second switch domain that comprises a fragment of FKBP or an analog thereof that has the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or a rapalog and is characterized by at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the FRB sequence of SEQ ID NO: 277 or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue therefrom. In one embodiment, the RCAR described herein comprises one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 275 (or SEQ ID NO: 276) and one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 277.

In some embodiments, the FKBP/FRB-based dimerization switch comprises a modified FRB-based switch domain that exhibits altered, e.g., improved, complex formation between the FRB-based switch domain, e.g., a modified FRB-based switch domain, the FKBP-based switch domain, and a dimerizing molecule, e.g., rapamycin or a rapalog, e.g., RAD001. In one embodiment, the modified FRB-based switch domain comprises one or more mutations, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected from mutations at amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105 and F2108, wherein the wild-type amino acid is mutated to any other naturally occurring amino acid. In one embodiment, the mutant FRB comprises a mutation at E2032, wherein E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), such as in SEQ ID NO: 278, or leucine (E2032L), such as in SEQ ID NO: 279. In one embodiment, the mutant FRB comprises a mutation at T2098, wherein T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), such as in SEQ ID NO: 280. In one embodiment, the mutant FRB comprises a mutation at E2032 and at T2098, wherein E2032 is mutated to is replaced by any amino acid, and wherein T2098 is mutated to any amino acid, such as in SEQ ID NO: 281. In one embodiment, the mutant FRB comprises the mutation E2032I and T2098L, such as in SEQ ID NO: 282. In one embodiment, the mutant FRB comprises the mutation E2032L and T2098L, such as in SEQ ID NO: 283.

Table 18. Illustrative mutant FRB characterized by increased affinity for a dimerizing molecule Mutant FRB Amino acid sequence SEQ ID NO: Mutant form E2032I ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 278 Mutant form E2032L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 279 Mutant form T2098L ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 280 Mutant form E2032, T2098 ILWHEMWHEGLXEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLXQAWDLYYHVFRRISKTS, wherein X is any amino acid residue 281 Mutant form E2032I, T2098L ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 282 Mutant form
E2032L, T2098L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 283

Other suitable dimerization switches include a GyrB-GyrB based dimerization switch, a gibberellin based dimerization switch, a tag/binding agent based dimerization switch, and a halogen tag/SNAP tag based dimerization switch. Such switches and suitable dimerizing molecules will be apparent to one of ordinary skill in the art given the teachings provided herein.

Dimerizing molecule

The association between the switch domains is facilitated by a dimerizing molecule. In the presence of the dimerizing molecule, the interaction or association between the switch domains provides for signal transmission between the polypeptide associated, for example, fused, with the first switch domain and the polypeptide associated, for example, fused, with the second switch domain. In the presence of non-limiting levels of the dimerizing molecule, signal transmission is enhanced by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 times, for example, when measured in the system described herein.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogs), such as RAD001, can be used as dimerizing molecules in the FKBP/FRB-based dimerization switch described herein. In one embodiment, the dimerizing molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus, and AP21967. Additional rapamycin analogs suitable for use with FKBP/FRB-based dimerization switches are further described in the section entitled "Types of Combination Therapies" or in the subsection entitled "Combination with a Low-Dose Immune Response-Enhanced MTOR Inhibitor."

Split CAR

In some embodiments, a cell expressing a CAR uses a split CAR. The split CAR approach is described in more detail in WO2014/055442 and WO2014/055657, incorporated herein by reference. Briefly, a split CAR system comprises a cell expressing a first CAR having a first antigen-binding domain and a costimulatory domain (e.g., 4-1BB), and the cell also expresses a second CAR having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3-zeta). When the cell encounters the first antigen, the costimulatory domain is activated and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell killing activity is triggered. Thus, a cell expressing a CAR is only fully activated in the presence of both antigens. In some embodiments, the first antigen-binding domain recognizes BCMA, such as comprising an antigen-binding domain as described herein, and the second antigen-binding domain recognizes an antigen expressed on acute myeloid leukemia cells, such as CD123, CLL-1, CD34, FLT3, or folate receptor beta. In some embodiments, the first antigen-binding domain recognizes BCMA, such as comprising an antigen-binding domain as described herein, and the second antigen-binding domain recognizes an antigen expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

Co-expression of CAR with other molecules or agents

Co-expression of a second CAR

In some embodiments, a cell expressing a CAR described herein may further comprise a second CAR, such as a second CAR that comprises a different antigen-binding domain, such as for the same target (e.g., CD19) or a different target (e.g., a target other than CD19, such as a target described herein). In some embodiments, a cell expressing a CAR comprises a first CAR that targets a first antigen and comprises an intracellular signaling domain that has a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain that has a primary signaling domain but not a costimulatory signaling domain. The placement of a costimulatory signaling domain, such as 4-1BB, CD28, CD27, OX-40, or ICOS, in a first CAR and a primary signaling domain, such as CD3-zeta, in a second CAR can restrict CAR activity to cells that express both targets. In some embodiments, a cell expressing a CAR comprises a first CAR that comprises an antigen-binding domain, a transmembrane domain, and a costimulatory domain, and a second CAR that targets another antigen and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In some embodiments, a cell expressing a CAR comprises a first CAR that comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets another antigen and comprises an antigen-binding domain for an antigen, a transmembrane domain, and a costimulatory signaling domain.

In some embodiments, a cell expressing a CAR comprises an XCAR as described herein and an inhibitory CAR. In some embodiments, the inhibitory CAR comprises an antigen-binding domain that binds to an antigen found on normal cells but not on cancer cells, such as normal cells that also express X. In some embodiments, the inhibitory CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can be the intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecules, MHC class II molecules, GAL9, adenosine, and TGF (e.g., TGF-beta).

In some embodiments, if a cell expressing a CAR comprises two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, a cell expressing a first and a second CAR may have an antigen-binding domain of the first CAR, for example, as a fragment, for example, an scFv, that does not associate with the antigen-binding domain of the second CAR, for example, the antigen-binding domain of the second CAR is VHH.

In some embodiments, the antigen-binding domain comprises single-domain antigen-binding (SDAB) molecules, including molecules in which the complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, variable domains of heavy chains, binding molecules naturally lacking light chains, individual domains derived from traditional 4-chain antibodies, engineered domains, and single-domain frameworks other than those derived from antibodies. SDAB molecules may be any molecule known in the art or any single-domain molecule that is later known. SDAB molecules may be derived from any species, including, but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. The term also encompasses naturally occurring single-domain antibody molecules from species other than Camelidae and sharks.

In some embodiments, the SDAB molecule may be derived from a variable region of an immunoglobulin found in fish, such as that derived from an immunoglobulin isotype known as novel antigen receptor (NAR) found in shark serum. Methods for producing single-domain molecules derived from the variable region of a NAR ("IgNAR") are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

In some embodiments, the SDAB molecule is a naturally occurring single-domain antigen-binding molecule known as a light chain-less heavy chain. Such single-domain molecules are disclosed, for example, in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, this variable domain derived from a light chain-less heavy chain molecule is referred to herein as a VHH or nanobody to distinguish it from the traditional four-chain immunoglobulin VH. Such a VHH molecule may be derived from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Species other than Camelidae may produce light chain-less heavy chain molecules; such VHHs are within the scope of the present invention.

SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, deimmunized, and/or generated in vitro (e.g., selected by phage display).

It has also been found that in cells having a plurality of membrane-embedded chimeric receptors comprising an antigen-binding domain, interactions between the antigen-binding domains of the receptors may be undesirable, for example, because they inhibit the ability of one or more antigen-binding domains to bind their cognate antigens. Accordingly, cells having first and second non-naturally occurring membrane-embedded chimeric receptors comprising antigen-binding domains for which such interactions are minimized are disclosed herein. Nucleic acids encoding first and second non-naturally occurring membrane-embedded chimeric receptors comprising antigen-binding domains for which such interactions are minimized are also disclosed herein, as well as methods for producing and using such cells and nucleic acids. In some embodiments, the antigen-binding domain of one of the first and second membrane-integrated non-naturally occurring chimeric receptors comprises an scFv and the other comprises a single VH domain, such as a single VH domain from a camelid, shark or lamprey, or a single VH domain derived from a human or mouse sequence.

In some embodiments, the composition described herein comprises a first and a second CAR, wherein the antigen-binding domain of one of the first and second CARs does not comprise a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen-binding domain of one of the first and second CARs is an scFv and the other is not an scFv. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises a single VH domain, such as a single camelid, shark or lamprey VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises a nanobody. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises a camelid VHH domain.

In some embodiments, the antigen-binding domain of one of the first and second CARs comprises an scFv and the other comprises a single VH domain, such as a single VH domain of camelids, sharks or lampreys, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises an scFv and the other comprises a nanobody. In some embodiments, the antigen-binding domain of one of the first and second CARs comprises an scFv and the other comprises a camelid VHH domain.

In some embodiments, the binding of the antigen-binding domain of the first CAR, when present on the cell surface, to its cognate antigen is not substantially reduced by the presence of the second CAR. In some embodiments, the binding of the antigen-binding domain of the first CAR to its cognate antigen in the presence of the second CAR is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99%, of the binding of the antigen-binding domain of the first CAR to its cognate antigen in the absence of the second CAR.

In some embodiments, the antigen-binding domains of the first and second CARs, when present on the cell surface, associate with each other to a lesser extent than if they were both scFv antigen-binding domains. In some embodiments, the antigen-binding domains of the first and second CARs associate with each other to a lesser extent, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% less, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% less, than if they were both scFv antigen-binding domains.

Co-expression of a CAR activity enhancing agent

In some embodiments, a CAR-expressing cell described herein may further express another agent, such as an agent that enhances the activity or fitness of the CAR-expressing cell.

For example, in some embodiments, the agent may be an agent that inhibits a molecule that modulates or regulates, e.g., inhibits, T cell function. In some embodiments, a molecule that modulates or regulates T cell function is an inhibitory molecule. Inhibitory molecules, e.g., PD1, in some embodiments may reduce the ability of CAR-expressing cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecule, MHC class II molecule, GAL9, adenosine, or TGF-beta.

In some embodiments, an agent, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., a siRNA or shRNA; or, e.g., an inhibitory protein or system, e.g., a clustered regularly interspaced short palindromic repeat (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used to inhibit the expression of a molecule that modulates or regulates, e.g., inhibits, T cell function in a cell expressing a CAR. In some embodiments, the agent is an shRNA, e.g., an shRNA described herein. In some embodiments, an agent that modulates or regulates, e.g., inhibits, T cell function is inhibited in a cell expressing a CAR. For example, a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, e.g., inhibits, T cell function is linked to a nucleic acid that encodes a component, e.g., all components, of a CAR.

In some embodiments, an agent that inhibits an inhibitory molecule comprises a first polypeptide, such as an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain as described herein. In some embodiments, the agent comprises a first polypeptide, e.g., from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, an MHC class I molecule, an MHC class II molecule, GAL9, adenosine, or TGF-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain as described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27, or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a signaling domain CD3-zeta as described herein). In some embodiments, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide that is an intracellular signaling domain as described herein (e.g., the CD28 signaling domain as described herein and/or the CD3-zeta signaling domain as described herein). PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two PD1 ligands, PD-L1 and PD-L2, have been shown to suppress T cell activation when bound to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is widely expressed in various human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

In some embodiments, an agent comprising an extracellular domain (ECD) of an inhibitory molecule, such as programmed cell death protein 1 (PD1), can be fused to a transmembrane domain and intracellular signaling domains, such as 4-1BB and CD3-zeta (also referred to herein as a PD1-based CAR). In some embodiments, a PD1-based CAR, when used in combinations with an XCAR described herein, improves T cell persistence. In some embodiments, the CAR is a PD1-based CAR comprising the extracellular domain of PD1 indicated by underlining in SEQ ID NO: 24. In some embodiments, a PD1-based CAR comprises the amino acid sequence of SEQ ID NO: 24.

In some embodiments, a PD1-based CAR comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the agent comprises a nucleic acid sequence encoding a PD1-based CAR, such as a PD1-based CAR described herein. In some embodiments, the nucleic acid sequence for a PD1-based CAR is set forth as SEQ ID NO: 23, with the PD1 ECD underlined.

In another example, in some embodiments, an agent that enhances the activity of a cell expressing a CAR can be a costimulatory molecule or a ligand of a costimulatory molecule. Examples of costimulatory molecules include an MHC class I molecule, BTLA, and a Toll-like receptor ligand, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Additional examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8-alpha, CD8-beta, IL2R-beta, IL2R-gamma, IL7R-alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and a ligand that specifically binds to CD83, such as those described herein. Examples of costimulatory molecule ligands include CD80, CD86, CD40L, ICOSL, CD70, OX40L, 4-1BBL, GITRL, and LIGHT. In some embodiments, the costimulatory molecule ligand is a ligand for a costimulatory molecule that is different from the costimulatory molecule whose domain is used in the CAR. In some embodiments, the costimulatory molecule ligand is a ligand for the same costimulatory molecule as the costimulatory molecule whose domain is used in the CAR. In some embodiments, the costimulatory molecule ligand is 4-1BBL. In one embodiment, the costimulatory molecule ligand is CD80 or CD86. In some embodiments, the costimulatory molecule ligand is CD70. In some embodiments, the CAR-expressing immune effector cell described herein may be further engineered to express one or more additional costimulatory molecules or costimulatory molecule ligands.

Co-expression of CAR with chemokine receptor

In some embodiments, a cell expressing a CAR, such as a cell expressing a CD19 CAR, described herein further comprises a chemokine receptor molecule. Transgenic expression of the chemokine receptors CCR2b or CXCR2 in T cells enhances their migration to solid tumors secreting CCL2 or CXCL1, including melanoma and neuroblastoma (Craddock et al., J Immunother . 2010 Oct; 33(8):780-8 and Kershaw et al., Hum Gene Ther . 2002 Nov 1; 13(16):1971-80). Thus, without being limited by theory, it is believed that chemokine receptors expressed on CAR-expressing cells that recognize chemokines secreted by tumors, such as solid tumors, can improve the homing of a CAR-expressing cell to a tumor, promote the infiltration of a CAR-expressing cell into a tumor, and enhance the antitumor efficacy of a CAR-expressing cell. The chemokine receptor molecule may comprise a naturally occurring or recombinant chemokine receptor or a chemokine-binding fragment thereof. A chemokine receptor molecule suitable for expression in a cell expressing a CAR (e.g., a CAR-Tx) described herein includes a CXC chemokine receptor (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3C chemokine receptor (e.g., CX3CR1), a XC chemokine receptor (e.g., XCR1), or a chemokine-binding fragment thereof. In some embodiments, a chemokine receptor molecule to be expressed in conjunction with a CAR described herein is selected based on the chemokine(s) secreted by the tumor. In some embodiments, a cell expressing a CAR described herein further comprises, e.g., expresses, a CCR2b receptor or a CXCR2 receptor. In some embodiments, a CAR described herein and a chemokine receptor molecule are in the same vector or are in two different vectors. In some embodiments in which a CAR described herein and a chemokine receptor molecule are in the same vector, the CAR and the chemokine receptor molecule are under the control of two different promoters or are under the control of the same promoter.

Nucleic acid constructs encoding CAR

The present invention also provides an immune effector cell, such as one produced by a method described herein, that comprises a nucleic acid molecule encoding one or more CAR constructs described herein. In some embodiments, the nucleic acid molecule is in the form of a transcript that is messenger RNA. In some embodiments, the nucleic acid molecule is in the form of a DNA construct.

The nucleic acid molecules described herein may be a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, the nucleic acid molecule is mRNA encoding a CAR polypeptide described herein. In other embodiments, the nucleic acid molecule is a vector that contains any of the above nucleic acid molecules.

In some embodiments, the antigen-binding domain of a CAR of the invention (e.g., an scFv) is encoded by a nucleic acid molecule whose sequence has been codon-optimized for expression in a mammalian cell. In some embodiments, the entire CAR construct of the invention is encoded by a nucleic acid molecule whose entire sequence has been codon-optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons encoding the same amino acid) in the coding DNA is biased between species. Such codon degeneracy allows for the encoding of the identical polypeptide by different nucleotide sequences. Various methods for codon optimization are known in the art and include, for example, those disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

Accordingly, in some embodiments, an immune effector cell, such as one produced by a method described herein, comprises a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain that binds to a tumor antigen described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) comprising a stimulatory domain, such as a costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and/or a primary signaling domain (e.g., a primary signaling domain described herein, such as a zeta chain described herein).

The present invention also provides vectors into which a nucleic acid molecule encoding a CAR, such as a nucleic acid molecule as described herein, is inserted. Vectors derived from retroviruses, such as lentivirus, are suitable tools for achieving long-term gene transfer, since they provide for long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. The retroviral vector can also be, for example, a gamma-retroviral vector. A gamma retroviral vector may comprise, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, such as a gene encoding a CAR. A gamma retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen necrosis virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3(6): 677-713.

In some embodiments, the vector comprising a nucleic acid encoding the desired CAR is an adenovirus vector (A5/35). In some embodiments, expression of nucleic acids encoding CARs can be achieved using transposons such as sleeping beauty, CRISPR, CAS9, and zinc finger nucleases. See below, June et al. 2009, Nature Reviews Immunology 9.10: 704-716, incorporated herein by reference.

Briefly, expression of natural or synthetic CAR-encoding nucleic acids is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or portion thereof to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

Nucleic acid can be cloned into a variety of vector types. For example, nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe vectors, and sequencing vectors.

In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, New York, and other textbooks on virology and molecular biology. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector comprises an origin of replication functional in at least one organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers (e.g., from WO 01/96584; WO 01/29058 and U.S. Patent No. 6,326,193).

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. They are typically located in a region 30-110 bp upstream of the transcription start site, although a number of promoters have been shown to also contain functional elements downstream of the transcription start site. The spacing between promoter elements is often flexible, so that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) gene promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function together or independently to activate transcription. Exemplary promoters include those of the CMV IE, EF-1α, ubiquitin C, or phosphoglycerate kinase (PGK) genes.

An example of a promoter that is capable of driving expression of a nucleic acid molecule encoding a CAR in a mammalian T cell is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to efficiently drive expression of CAR from nucleic acid molecules cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In some embodiments, the EF1a promoter comprises a sequence as set forth in the examples.

Another example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving the expression of any polynucleotide sequence operably linked to it at high levels. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the murine mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin gene promoter, the myosin gene promoter, the elongation factor-1α gene promoter, the hemoglobin gene promoter, and the creatine kinase gene promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on the expression of a polynucleotide sequence to which it is operably linked if such expression is desired, or turning off the expression if expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein-inducible promoter, a glucocorticoid-inducible promoter, a progesterone-inducible promoter, and a tetracycline-inducible promoter.

Another example of a promoter is the phosphoglycerate kinase (PGK) gene promoter. In some embodiments, a truncated PGK gene promoter may be desired (e.g., a PGK gene promoter with one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotide deletions compared to the wild-type PGK gene promoter sequence).

The nucleotide sequences of illustrative PGK gene promoters are shown below.

PGK WT gene promoter:

ACCCCTCTCTCCAGCCACTAAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGG CGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGC ACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGC CTGCACTTCTTACACGCTCTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGGTGCGGGTCTCGTCGGCGCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGGTTGGGGTTGGGGCACCATAAGCT (SEQ ID NO: 190)

Illustrative truncated PGK gene promoters:

PGK100:

ACCCCTCTCTCCAGCCACTAAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTG (SEQ ID NO: 198)

PGK200:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGACGGTAACG (SEQ ID NO: 191)

PGK300:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGC GCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCG (SEQ ID NO: 192)

PGK400:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGC GACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGAACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCG (SEQ ID NO: 193)

The vector may also contain, for example, a secretion promoting signal sequence, a polyadenylation signal and a transcription terminator (e.g. from the bovine growth hormone (BGH) gene), an element enabling episomal and prokaryotic replication (e.g. the SV40 and ColE1 replication origin or others known in the art), and/or elements enabling selection (e.g. an ampicillin resistance gene and/or a zeocin resistance marker).

To assess the expression of the CAR polypeptide or portions thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells that were sought to be transfected or infected by the viral vectors. In some embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to ensure expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo, etc.

Reporter genes are used to identify potentially transfected cells and to assess the functional properties of regulatory sequences. Typically, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue and that encodes a polypeptide whose expression results in some readily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after introduction of the DNA into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared by known techniques or purchased commercially. Typically, the construct with the minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

In some embodiments, the vector may comprise two or more nucleic acid sequences encoding a CAR, such as a CAR described herein, such as a CD19 CAR, and a second CAR, such as an inhibitory CAR or a CAR that specifically binds to an antigen other than CD19. In such embodiments, the two or more nucleic acid sequences encoding the CAR are encoded by a single nucleic acid molecule in the same reading frame and as a single polypeptide chain. In some embodiments, the two or more CARs may, for example, be separated by one or more peptide cleavage sites (e.g., a self-cleavage site or a substrate for an intracellular protease). Examples of peptide cleavage sites include T2A, P2A, E2A, or F2A sites.

Methods for introducing genes into a cell and providing their expression in it are known in the art. As for the expression vector, the vector can be easily introduced into the host cell, for example, a mammalian cell, a bacterium, a yeast or an insect, by any method, for example, a method known in the art. For example, the expression vector can be transferred into the host cell by a physical, chemical or biological method.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, New York. A suitable method for introducing a polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as humans. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersed systems such as macromolecular complexes, nanocapsules, microspheres, granules and lipid systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods for targeted delivery of nucleic acids are available from the art, such as delivery of polynucleotides using targeting nanoparticles or another suitable submicron delivery system.

In case of using a delivery system other than a virus, an exemplary delivery vehicle is a liposome. For introduction of nucleic acids into a host cell (in vitro, ex vivo or in vivo) use of lipid formulations is contemplated. In some embodiments, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, placed in a lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector based association compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles or in a "squeezed" structure. They may also simply be placed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contains long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

Suitable lipids can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of unilamellar and multilamellar lipid carriers formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilamellar liposomes have several lipid layers separated by an aqueous environment. They are formed spontaneously by suspending phospholipids in excess aqueous solution. The lipid components undergo self-reorganization before forming closed structures and entrap water and solutes between the lipid bilayers (Ghosh et al., 1991, Glycobiology 5: 505-10). However, compositions that have structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also proposed.

In order to confirm the presence of a recombinant nucleic acid sequence in a host cell, various assays can be carried out, regardless of the method used to introduce exogenous nucleic acids into the host cell or otherwise expose the cell to an inhibitor of the present invention. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological methods (ELISA and Western blotting) or by the assays described herein, to identify agents within the scope of the present invention.

RNA transfection

Disclosed herein are methods for producing in vitro transcribed RNA encoding a CAR. CAR encoding RNA and methods for using the CAR are described, for example, in paragraphs 553-570 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

The immune effector cell may comprise a CAR encoded by messenger RNA (mRNA). In some embodiments, mRNA encoding a CAR described herein is introduced into an immune effector cell, such as one produced by a method described herein, to produce a cell expressing the CAR.

In some embodiments, in vitro transcribed RNA encoding a CAR can be introduced into a cell in the form of transient transfection. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, a synthetic DNA sequence, or any other suitable source of DNA. The desired template for in vitro transcription is a CAR as described herein. For example, a template for RNA encoding a CAR comprises an extracellular region comprising a single-chain variable domain of an antibody to a tumor-associated antigen as described herein; a hinge region (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein, such as a CD8a transmembrane domain), and a cytoplasmic region that contains an intracellular signaling domain, e.g., an intracellular signaling domain described herein, e.g., comprising a CD3-zeta signaling domain and a 4-1BB signaling domain.

In some embodiments, the DNA to be used in the PCR comprises an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In some embodiments, the nucleic acid may comprise some or all of the 5' and/or 3' untranslated regions (UTRs). The nucleic acid may comprise exons and introns. In some embodiments, the DNA to be used in the PCR is a human nucleic acid sequence. In some embodiments, the DNA to be used in the PCR is a human nucleic acid sequence comprising a 5' and 3' UTR. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that comprises portions of genes ligated together to form an open reading frame that encodes a fusion protein. The DNA pieces ligated together may be from one organism or from more than one organism.

PCR is used to produce a template for in vitro transcription of mRNA that is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of DNA to be used as a template for PCR. As used herein, the term "substantially complementary" refers to nucleotide sequences wherein most or all of the bases in the primer sequence are complementary or one or more bases are non-complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the intended target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the template DNA. For example, primers can be designed to amplify a portion of a nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTR. Primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In some embodiments, primers are designed to amplify a coding region of human cDNA, including all or portions of the 5' and 3' UTR. Primers useful for PCR can be prepared by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides in the template DNA that are located upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a location in the 5' direction from the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain regions of nucleotides that are substantially complementary to nucleotides in the double-stranded DNA template that are located downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a location in the 3' direction from the DNA sequence to be amplified relative to the coding strand.

The methods disclosed herein may employ any DNA polymerase suitable for PCR. The reagents and polymerase are commercially available from a number of sources.

Chemical structures that can promote stability and/or translation efficiency can also be used. The RNA in some embodiments comprises a 5' and 3' UTR. In some embodiments, the length of the 5' UTR is from one to 3000 nucleotides. The length of the 5' and 3' UTR sequences to be added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the length of the 5' and 3' UTR as needed to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs may be naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest may be added by incorporating UTR sequences into the forward and reverse primers or by any other modifications to the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest may be useful for modifying the stability and/or translation efficiency of RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease mRNA stability. Therefore, 3' UTRs may be selected or designed to increase the stability of transcribed RNA based on the properties of UTRs that are well known in the art.

In some embodiments, the 5' UTR may comprise a Kozak sequence from an endogenous nucleic acid. Alternatively, if a 5' UTR that is not endogenous to the nucleic acid of interest is added by PCR as described above, the Kozak consensus sequence may be modified by adding the 5' UTR sequence. Kozak sequences may increase the translation efficiency of some RNA transcripts, but do not appear to be necessary to ensure efficient translation of all RNAs. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR may be the 5' UTR from an RNA virus whose RNA genome is stable in cells. In other embodiments, different nucleotide analogs in the 3' or 5' UTR may be used to prevent mRNA from being destroyed by exonucleases.

To allow RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be added to the DNA template upstream of the sequence to be transcribed. If a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the promoter for RNA polymerase is included in the PCR product upstream of the open reading frame to be transcribed. In some embodiments, the promoter is a promoter for T7 polymerase, as described elsewhere herein. Other useful promoters include, but are not limited to, promoters for T3 and SP6 RNA polymerases. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

In some embodiments, the mRNA contains both a 5' cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatemeric product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end results in normal-sized mRNA that is not effective for transfection in eukaryotes, even if polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The traditional method of integrating poly(A)/(T) stretches into DNA template is molecular cloning. However, the poly(A)/(T) sequence integrated into plasmid DNA may cause plasmid instability, as a result of which plasmid DNA templates obtained from bacterial cells are often heavily contaminated with deletions and other aberrations. This results in cloning procedures being not only laborious and time-consuming, but also often unreliable. Therefore, there is a great need for a method that allows the construction of DNA templates with poly(A)/(T) stretches at the 3' end without cloning.

The poly(A)/(T) segment in the transcription DNA template can be obtained by PCR using a reverse primer containing a poly(T) tail, such as a 100 T tail (SEQ ID NO: 31) (the size can be 50-5000 T (SEQ ID NO: 32)) or after PCR using any other method, including, but not limited to, in vitro DNA ligation or recombination. Poly(A) tails also provide RNA stability and reduce their degradation. Typically, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In some embodiments, the length of the poly(A) tail is from 100 to 5000 adenosine residues (e.g., SEQ ID NO: 33).

Poly(A) tails of RNA can be further extended after in vitro transcription by using a poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP). In some embodiments, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 34) results in an approximately two-fold increase in RNA translation efficiency. Additionally, addition of various chemical groups to the 3' end can increase mRNA stability. Such addition can include addition of modified/artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase RNA stability.

5' cap structures also provide stability to RNA molecules. In some embodiments, RNAs produced by the methods disclosed herein contain a 5' cap. The presence of a 5' cap is achieved using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

RNAs produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solutes suitable for cell electroporation may be included, which may include factors that promote cell entry and cell viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be introduced into target cells by any of a number of different methods, such as commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg, Germany)), cationic liposome-mediated transfection using lipofection, encapsulation in polymers, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

Non-viral delivery methods

In some embodiments, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or organism of a subject.

In some embodiments, the non-viral method includes using a transposon (also called a mobile genetic element). In some embodiments, a transposon is a DNA fragment that can self-insert itself into a specific location in the genome, such as a DNA fragment that is capable of self-replication and inserting a copy of itself into the genome, or a DNA fragment that can be cut from a longer nucleic acid by splicing and inserted into another location in the genome. For example, a transposon comprises a DNA sequence consisting of inverted repeats flanking genes for transposition.

Exemplary transposon-based nucleic acid delivery methods include the sleeping beauty transposon system (SBTS) and the piggyBac™ (PB) transposon system. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14–20; Singh et al. Cancer Res. 15(2008):2961–2971; Huang et al. Mol. Ther. 16(2008):580–589; Grabundzija et al. Mol. Ther. 18(2010):1200–1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515–16; Bell et al. Nat. Protoc. 2.12(2007):3153–65; and Ding et al. Cell. 122.3(2005):473–83, all of which are incorporated herein by reference.

SBTS contains two components: 1) a transposon containing a transgene, and 2) a source of the transposase enzyme. The transposase can mediate the transfer of a transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome. For example, the transposase binds to the carrier/donor DNA, excises the transposon (including the transgene(s)) from the plasmid, and inserts it into the host cell genome. See, e.g., Aronovich et al. higher.

Exemplary transposons include the pT2-based transposon. See, e.g., Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47 and Singh et al. Cancer Res. 68.8(2008):2961-2971, all of which are incorporated herein by reference. Exemplary transposases include a Tc1/mariner-type transposase, such as the SB10 transposase or the SB11 transposase (a hyperactive transposase that can be expressed, for example, under the control of the cytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al. and Grabundzija et al., all of which are incorporated herein by reference.

The use of SBTS enables efficient integration and expression of a transgene, such as a nucleic acid encoding a CAR described herein. The present document provides methods for producing a cell, such as a T cell or NK cell, that stably expresses a CAR described herein, such as using a transposon system such as SBTS.

According to the methods described herein, in some embodiments, one or more nucleic acids, e.g., plasmids containing SBTS components, are delivered to a cell (e.g., a T or NK cell). For example, the nucleic acid(s) are(are) delivered using standard nucleic acid (e.g., plasmid DNA) delivery methods, such as those described herein, such as electroporation, transfection, or lipofection. In some embodiments, the nucleic acid comprises a transposon comprising a transgene, such as a nucleic acid encoding a CAR as described herein. In some embodiments, the nucleic acid comprises a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR as described herein), and a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a two nucleic acid system is provided, such as a two plasmid system, such as where the first plasmid contains a transposon containing a transgene and the second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-delivered to the host cell.

In some embodiments, cells, such as T or NK cells, that express a CAR described herein are produced by a combination of gene insertion using SBTS and genetic editing using a nuclease (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, or engineered meganucleases that are reshaped homing endonucleases).

In some embodiments, the use of a non-viral delivery method allows for reprogramming of cells, such as T or NK cells, and direct infusion of the cells into a subject. Advantages of non-viral vectors include, but are not limited to, ease and relatively low cost of producing sufficient quantities to meet the requirements of a patient population, stability upon storage, and lack of immunogenicity.

Methods of production/obtaining

The present invention also provides methods for making a cell as disclosed herein, such as methods for engineering a T cell or NK cell to express a nucleic acid molecule encoding one or more CAR constructs as disclosed herein. In some embodiments, the manufacturing methods disclosed herein are used to make a cell comprising a nucleic acid molecule encoding two CARs as disclosed herein (e.g., a BCMA CAR and a CD19 CAR as disclosed herein). In some embodiments, the manufacturing methods disclosed herein are used to make a cell comprising a nucleic acid molecule encoding a CAR in diabody format as disclosed herein, such as a BCMA/CD19 CAR in diabody format as disclosed herein. In some embodiments, the manufacturing methods disclosed herein are used to manufacture a cell comprising two nucleic acid molecules, each encoding a CAR disclosed herein (e.g., one nucleic acid molecule encoding a CAR for BCMA and one nucleic acid molecule encoding a CAR for CD19). In some embodiments, provided herein is a population of cells (e.g., immune effector cells, such as T cells or NK cells) produced by any of the manufacturing methods described herein.

Method using activation

In some embodiments, the methods disclosed herein can produce immune effector cells engineered to express one or more CARs in less than 24 hours. Without being bound by theory, it is believed that the methods provided herein allow the undifferentiated phenotype of T cells, such as naive T cells, to be maintained during the manufacturing process. Such CAR-expressing cells with an undifferentiated phenotype can persist longer and/or expand better in vivo after infusion. In some embodiments, CAR T cells produced by the manufacturing methods provided herein contain a higher percentage of memory T cells that are stem cells compared to CAR T cells produced by a traditional manufacturing process, such as as measured by scRNA-seq (e.g., as measured by the methods described in Example 7 with respect to Fig. 25A). In some embodiments, CAR-T cells produced by the manufacturing methods provided herein comprise a higher percentage of effector T cells compared to CAR-T cells produced by a traditional manufacturing method, such as as measured by scRNA-seq (e.g., as measured by the methods described in Example 7 with respect to Fig. 25B). In some embodiments, CAR-T cells produced by the manufacturing methods provided herein better retain T cell stemness compared to CAR-T cells produced by a traditional manufacturing method, such as as measured by scRNA-seq (e.g., as measured by the methods described in Example 7 with respect to Fig. 25C). In some embodiments, CAR-T cells produced by the manufacturing methods provided herein exhibit a lower level of hypoxia compared to CAR-T cells produced by a traditional manufacturing method, such as as measured by scRNA-seq (e.g., as measured by the methods described in Example 7 with respect to Fig. 25D). In some embodiments, CAR-T cells produced by the manufacturing methods provided herein exhibit a lower level of autophagy compared to CAR-T cells produced by a traditional manufacturing method, such as as measured by scRNA-seq (e.g., as measured by the methods described in Example 7 with respect to Fig. 25E). In some embodiments, the immune effector cells are engineered to comprise a nucleic acid molecule encoding two CARs disclosed herein (e.g., a BCMA CAR and a CD19 CAR disclosed herein). In some embodiments, the immune effector cells are engineered to comprise a nucleic acid molecule encoding a CAR in a diabody format as disclosed herein, such as a CAR for BCMA/CD19 in a diabody format as disclosed herein. In some embodiments, the immune effector cells are engineered to comprise two nucleic acid molecules, each encoding a CAR as disclosed herein (e.g., one nucleic acid molecule encoding a CAR for BCMA and one nucleic acid molecule encoding a CAR for CD19).

In some embodiments, the methods disclosed herein do not involve the use of a bead, such as Dynabeads® (e.g., Dynabeads® with CD3/CD28 antibodies), and do not involve the step of removing the beads. In some embodiments, CAR T cells made by the methods disclosed herein can be administered to a subject with minimal ex vivo expansion, such as in less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or without ex vivo expansion. Accordingly, the methods described herein provide a rapid manufacturing method for producing improved CAR-expressing cell products for use in treating a disease in a subject.

In some embodiments, the present invention provides methods for producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR) (e.g., one or more CARs, e.g., two CARs) comprising (i) contacting the population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates the CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting a population of cells (e.g., T cells) with a nucleic acid molecule(s) (e.g., a DNA or RNA molecule) encoding a CAR, thereby producing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (iii) collecting the population of cells (e.g., T cells) for storage (e.g., reconstituting the population of cells in a cryopreservation medium) or administration, wherein (a) step (ii) is performed in conjunction with step (i) or no later than 20 hours after the start of step (i), such as no later than 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), such as no later than 18 hours after the start of step (i), and step (iii) is performed no later than 26 hours after the start of step (i), such as no later than 22, 23, or 24 hours after the start of step (i), for example, not later than 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or not later than 20 hours after the start of step (i), for example not later than 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example not later than 18 hours after the start of step (i), and step (iii) is carried out not later than 30 hours after the start of step (ii), for example not later than 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the start of step (ii); or (c) the population of cells from step (iii) is not expanded or is expanded by an increase in number of no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of live cells, compared to the population of cells at the beginning of step (i). In some embodiments, the nucleic acid molecule of step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is in a viral vector, such as a viral vector selected from a lentiviral vector, an adenoviral vector or a retroviral vector. In some embodiments, the nucleic acid molecule of step (ii) is in a non-viral vector. In some embodiments, the nucleic acid molecule of step (ii) is in a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not in any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector(s) containing a nucleic acid molecule encoding a CAR.

In some embodiments, a population of cells (e.g., T cells) is collected from a sample obtained by apheresis (e.g., a sample obtained by leukapheresis) from a subject.

In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed and T cells (e.g., CD4+ T cells and/or CD8+ T cells) are isolated from the apheresis sample, such as using a cell sorting device (e.g., a CliniMACS® Prodigy® device). The isolated T cells (e.g., CD4+ T cells and/or CD8+ T cells) are then seeded to manufacture CAR-T using the activation-based method described herein. In some embodiments, selected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are subjected to one or more freeze-thaw cycles prior to seeding to make CAR-T.

In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and transported as a fresh product (e.g., a non-frozen product) to a cell manufacturing facility. T cells (e.g., CD4+ T cells and/or CD8+ T cells) are selected from the apheresis sample, such as using a cell sorting device (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are then seeded to make CAR-T using the activation-based method described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are subjected to one or more freeze-thaw cycles prior to seeding to make CAR-T.

In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. T cells (e.g., CD4+ T cells and/or CD8+ T cells) are collected from the apheresis sample, such as using a cell sorting device (e.g., a CliniMACS® Prodigy® device). The collected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are then transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The collected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are subsequently thawed and seeded to manufacture CAR-T using the activation-based method described herein.

In some embodiments, cells (e.g., T cells) are contacted with anti-CD3 antibodies and anti-CD28 antibodies for, e.g., 12 hours, followed by transduction with a vector (e.g., a lentiviral vector) (e.g., one or more vectors) encoding a CAR (e.g., one or more CARs). After 24 hours of culture, the cells are washed and formulated for storage or administration.

Without being bound by theory, it is believed that short-term stimulation of CD3 and CD28 may facilitate efficient transduction of self-renewing T cells. Compared to traditional approaches to CAR-T fabrication, the activation-based method provided herein does not require prolonged ex vivo expansion. Similar to the cytokine-based method, the activation-based method provided herein also preserves undifferentiated T cells during CAR-T fabrication.

In some embodiments, the cell population is contacted with an agent that stimulates the CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the cell surface.

In some embodiments, an agent that stimulates a CD3/TCR complex is an agent that stimulates CD3. In some embodiments, an agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, an agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, an agent that stimulates a CD3/TCR complex is selected from an antibody (e.g., a single domain antibody (e.g., a single heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates the CD3/TCR complex is an antibody. In some embodiments, the agent that stimulates the CD3/TCR complex is an anti-CD3 antibody. In some embodiments, the agent that stimulates the costimulatory molecule is selected from an antibody (e.g., a single domain antibody (e.g., a single heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates the costimulatory molecule is an antibody. In some embodiments, the agent that stimulates the costimulatory molecule is an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3/TCR complex or the agent that stimulates the costimulatory molecule does not comprise a bead. In some embodiments, the agent that stimulates the CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates the costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates the CD3/TCR complex and the agent that stimulates the costimulatory molecule comprise TransAct™ for T cells.

In some embodiments, the matrix comprises a polymeric, such as biodegradable or biocompatible, inert material, such as one that is non-toxic to cells, or consists of such a material. In some embodiments, the matrix consists of hydrophilic polymer chains that achieve maximum mobility in an aqueous solution due to chain hydration. In some embodiments, the mobile matrix can consist of collagen-based compositions, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or an extracellular matrix. The polysaccharide can include, for example, cellulose ethers, starch, acacia, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum, or alginate. Other polymers may include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimines, polyquaternium polymers, polyphosphazenes, polyvinyl alcohols, polyvinyl acetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the mobile matrix is a dextran polymer.

In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs).

In some embodiments, when co-transducing with two nucleic acid molecules (e.g., lentiviral vectors) each encoding a CAR disclosed herein (e.g., one nucleic acid molecule encoding a CAR for BCMA and one nucleic acid molecule encoding a CAR for CD19, disclosed herein), each of the vectors containing the nucleic acid molecules encoding the CARs can be added to a reaction mixture (e.g., containing a population of cells) at a different multiplicity of infection (MOI).

Without being limited by theory, it is believed that in some embodiments, using different MOIs for vectors containing nucleic acid molecules that encode different CAR molecules can affect the final composition of the cell population. For example, in the case of cotransduction with a lentiviral vector encoding a CAR for BCMA and a lentiviral vector encoding a CAR for CD19, different MOIs can be used to maximize the percentage of mono-BCMA CAR T cells and BCMA/CD19 dual-specific CAR T cells while producing fewer mono-CD19 CAR T cells and untransduced cells.

In some embodiments, in the case of cotransduction with a lentiviral vector encoding a CAR for BCMA and a lentiviral vector encoding a CAR for CD19, the population of cells is contacted with the first viral vector at a multiplicity of infection (MOI) that is greater than, equal to, or less than the MOI at which the population of cells is contacted with the second viral vector. In some embodiments, the population of cells is contacted with the first viral vector at a multiplicity of infection (MOI) that is greater than the MOI at which the population of cells is contacted with the second viral vector.

In some embodiments, the population of cells is contacted with a first viral vector at a first MOI and with a second viral vector at a second MOI, such that the resulting population of cells comprises a first population of cells that contain a CAR for BCMA but not a CAR for CD19, a second population of cells that contain a CAR for CD19 but not a CAR for BCMA, and a third population of cells that contain both a CAR for BCMA and a CAR for CD19, where:

(a) the total number of viable cells in the combined second and third populations is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10;

(b) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations, e.g., when determined by the method described in Example 10;

(c) the total number of viable cells in the combined first and third populations is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the resulting population, e.g., when determined by the method described in Example 10;

(d) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10; or

(e) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the second population, e.g., when determined by the method described in Example 10.

In some embodiments, the population of cells is contacted with a second viral vector at an MOI, such as an MOI that is sufficiently lower than the MOI at which the population of cells is contacted with the first viral vector, such that the resulting population of cells:

(a) the total number of viable cells in the combined second and third populations is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10;

(b) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations, e.g., when determined by the method described in Example 10;

(c) the total number of viable cells in the combined first and third populations is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the resulting population, e.g., when determined by the method described in Example 10;

(d) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10; or

(e) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the second population, e.g., when determined by the method described in Example 10.

In some embodiments, the population of cells is contacted with a first viral vector at a first MOI and the population of cells is contacted with a second viral vector at a second MOI, such that the resulting population of cells comprises:

(a) the total number of viable cells in the combined second and third populations is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10;

(b) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations, e.g., when determined by the method described in Example 10;

(c) the total number of viable cells in the combined first and third populations is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the resulting population, e.g., when determined by the method described in Example 10;

(d) the total number of viable cells in the second population is less than or equal to about 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations, e.g., when determined by the method described in Example 10; or

(e) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the second population, e.g., when determined by the method described in Example 10.

In some embodiments, the population of cells is contacted with

(a) a first viral vector at an MOI of from about 1 to about 10 (e.g., from about 2 to about 9, from about 3 to about 8, from about 4 to about 7, from about 5 to about 6, from about 1 to about 8, from about 1 to about 6, from about 1 to about 4, from about 8 to about 10, from about 6 to about 10, from about 4 to about 10, from about 1 to about 3, from about 2 to about 4, from about 3 to about 5, from about 4 to about 6, from about 5 to about 7, from about 6 to about 8, from about 7 to about 9, from about 8 to about 10, from about 2.5 to about 5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10);

(b) a second viral vector at an MOI of from about 0.1 to about 5 (e.g., from about 0.2 to about 4, from about 0.3 to about 3, from about 0.4 to about 2, from about 0.5 to about 1, from about 0.6 to about 0.9, from about 0.7 to about 0.8, from about 0.1 to about 4, from about 0.1 to about 3, from about 0.1 to about 2, from about 0.1 to about 1, from about 0.1 to about 0.5, from about 4 to about 5, from about 3 to about 5, from about 2 to about 5, from about 1 to about 5, from about 0.5 to about 5, from about 0.2 to about 5, from about 0.1 to about 0.5, from about 0.2 to about 1, from about 0.5 up to about 2, from about 1 to about 3, from about 2 to about 4, from about 3 to about 5, from about 0.5 to about 1, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5);

(c) a first viral vector at an MOI that is at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the MOI at which the population of cells is contacted with a second viral vector, or at least about 1-fold (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100-fold, such as about 2 to about 50-fold, about 3 to 20-fold, about 5 to about 15-fold, or about 8 to about 10-fold) greater therefrom; and/or

(d) a second viral vector at an MOI that is no more than 1/X, where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100, of the MOI at which the cell population is contacted with the first viral vector.

In some embodiments, the population of cells is contacted with the first viral vector at an MOI of about 2.5 to about 5. In some embodiments, the population of cells is contacted with the second viral vector at an MOI of about 0.5 to about 1.0. In some embodiments, the MOI for the first viral vector is about 8 to about 10 times higher than the MOI at which the population of cells is contacted with the second viral vector. In some embodiments, the MOI for the second viral vector is no more than 1/X, where X is 6, 8, 10, or 12, of the MOI at which the population of cells is contacted with the first viral vector.

In some embodiments, in step (ii), the population of cells is contacted with:

(a) a first viral vector at an MOI of from about 4 to about 5 (e.g., about 4.75); and/or

(b) a second viral vector at an MOI of from about 0.2 to about 1 (e.g., about 0.5).

In some embodiments, in step (ii), the population of cells comprises from about 1×10 ⁸ to about 5×10 ⁹ (e.g., from about 2×10 ⁸ to about 2×10 ⁹ or from about 4×10 ⁸ to about 1×10 ⁹ ) total viable cells. In some embodiments, the cells are suspended in culture at a concentration of from about 1×10 ⁶ to about 1×10 ⁷ (e.g., from about 2×10 ⁶ to about 5×10 ⁶ or from about 3×10 ⁶ to about 4×10 ⁶ ) viable cells/mL.

The precise MOI used for each vector can be adjusted or determined based on a number of factors, including, but not limited to, the properties of the viral vector batch, the characteristics of the cells to be transduced, and the transduction efficiency. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs simultaneously with contacting the cell population with an agent that stimulates the CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the cell surface, as described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the start of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the cell surface, as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 20 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 19 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 18 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 17 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 16 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 15 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 14 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 14 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 13 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 12 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 11 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 10 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 9 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 8 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 7 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the cell population with the nucleic acid molecule(s) encoding the CAR occurs no later than 6 hours after the initiation of contacting the cell population with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 5 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 4 hours after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, contacting the population of cells with a nucleic acid molecule(s) encoding a CAR occurs no later than 3 hours after the start of contacting the population of cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of cells, as described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 2 hours after the start of contacting the population of cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of cells, as described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 1 hour after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR occurs no later than 30 minutes after the start of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above.

In some embodiments, the cell population is collected for storage or administration.

In some embodiments, the population of cells is collected for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is collected for storage or administration no later than 26 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is collected for storage or administration no later than 25 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of cells described above. In some embodiments, the population of cells is collected for storage or administration no later than 24 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of cells described above. In some embodiments, the population of cells is collected for storage or administration no later than 23 hours after the initiation of contacting the population of cells with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the surface of cells described above. In some embodiments, the cell population is collected for storage or administration no later than 22 hours after the cell population is first exposed to the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above.

In some embodiments, the cell population is not expanded ex vivo.

In some embodiments, the cell population is expanded to an increase in number of no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, such as as assessed by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the cell population is expanded by no more than 5% increase in number, such as as measured by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface, as described above. In some embodiments, the cell population is expanded by no more than 10% increase in number, such as as measured by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface, as described above. In some embodiments, the cell population is expanded by no more than 15% increase in number, such as as measured by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface, as described above. In some embodiments, the cell population is expanded by no more than 20% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface, as described above. In some embodiments, the cell population is expanded by no more than 25% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface, as described above. In some embodiments, the cell population is expanded by no more than 30% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface, as described above. In some embodiments, the cell population is expanded to an increase in number of no more than 35%, for example, as measured by the number of living cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above. In some embodiments, the cell population is expanded to an increase in number of no more than 40%, for example, as measured by the number of living cells, compared to the cell population before it was contacted with the agent that stimulates the CD3/TCR complex and/or the agent that stimulates the costimulatory molecule on the cell surface described above.

In some embodiments, the cell population is expanded for no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, such as as assessed by the number of living cells, compared to the cell population before it was exposed to one or more cytokines described above.

In some embodiments, the method using activation is performed in serum-free cell media. In some embodiments, the method using activation is performed in cell media containing one or more cytokines selected from IL-2, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) or IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, hetIL-15 comprises the amino acid sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQG (SEQ ID NO: 309). In some embodiments, hetIL-15 comprises an amino acid sequence having at least about 70, 75, 80, 85, 90, 95, or 99% identity to SEQ ID NO: 309. In some embodiments, the method using activation is performed in cell media comprising an LSD1 inhibitor. In some embodiments, the method using activation is performed in cell media comprising a MALT1 inhibitor. In some embodiments, the serum-free cell media comprises a serum substitute. In some embodiments, the serum substitute is CTS™ Immune Cell Serum Replacer (ICSR). In some embodiments, the ICSR level can be, for example, up to 5%, such as about 1%, 2%, 3%, 4%, or 5%. Without being limited by theory, it is believed that the use of cell media, such as the rapid media shown in Table 21 or Table 25, containing ICSR, such as 2% ICSR, may provide improved cell viability during the manufacturing process described herein.

In some embodiments, the present invention provides methods for producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), comprising (a) obtaining an apheresis sample (e.g., a fresh or cryopreserved leukapheresis sample) collected from a subject; (b) selecting T cells from the apheresis sample (e.g., using negative selection, positive selection, or bead-free selection); (c) seeding the isolated T cells at, e.g., 1×10 ⁶ to 1×0 ⁷ cells/mL; (d) contacting the T cells with an agent that stimulates the T cells, such as an agent that stimulates the CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells (e.g. contacting the T cells with an anti-CD3 antibody and/or an anti-CD28 antibody, such as contacting the T cells with TransAct); (e) contacting the T cells with a nucleic acid molecule(s) (e.g. a DNA or RNA molecule) encoding the CAR (e.g. contacting the T cells with a virus containing a nucleic acid molecule(s) encoding the CAR) for, such as 6-48 hours, such as 20-28 hours; and (f) washing and collecting the T cells for storage (e.g. reconstitution of the T cells in cryopreservation medium) or administration. In some embodiments, step (f) is performed no later than 30 hours after the start of step (d) or (e), such as no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (d) or (e).

In some embodiments, provided herein is a population of cells (e.g., immune effector cells, such as T cells or NK cells) produced by any of the manufacturing methods described herein (e.g., a method using activation described herein).

In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in the cell population at the end of the method of manufacture (e.g., at the end of the cytokine-using method or the activation-using method described herein) (1) is the same, (2) is different, e.g., by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is increased, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, compared to the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ cells, in a population of cells at the start of a manufacturing method (e.g., at the start of a cytokine-based method or an activation-based method described herein). In some embodiments, the cell population at the end of the manufacturing method (e.g., the end of the cytokine-based method or the activation-based method described herein) exhibits a higher percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher), compared to cells obtained by an otherwise similar method that lasts, e.g., more than 26 hours (e.g., lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or that involves expanding the cell population in vitro for, for example, more than 3 days (e.g., expansion of a cell population in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+ CD45RO- CCR7+ T cells, in the cell population at the end of the manufacturing method (e.g., at the end of the cytokine-based method or the activation-based method described herein) is at least 20, 25, 30, 35, 40, 45, 50, 55, or 60%.

In some embodiments, the percentage of central memory cells, e.g. central memory T cells, e.g. CD95+ central memory T cells, in the cell population at the end of the method of manufacture (e.g., at the end of the method using cytokines or the method using activation described herein) (1) is the same, (2) is different, e.g., by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is reduced, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, compared to the percentage of central memory cells, e.g. central T cells memory, such as CD95+ central memory T cells, in a population of cells at the start of the manufacturing method (e.g., at the start of the cytokine-based method or the activation-based method described herein). In some embodiments, the cell population at the end of the method of manufacture (e.g., the end of the cytokine-based method or the activation-based method described herein) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower), compared to cells obtained by an otherwise similar method that lasts, e.g., more than 26 hours (e.g., lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or that involves expanding the cell population in vitro for, e.g., more than 3 days (e.g., expanding a cell population in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

In some embodiments, the percentage of central memory cells, such as central memory T cells, such as central memory CD95+ T cells, in the cell population at the end of the manufacturing method (e.g., at the end of the cytokine-based method or the activation-based method described herein) is no more than 40, 45, 50, 55, 60, 65, 70, 75, or 80%.

In some embodiments, the cell population at the end of the method of manufacture (e.g., at the end of the cytokine-based method or the activation-based method described herein) after in vivo administration persists longer or expands at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) than cells produced by an otherwise similar method that lasts, e.g., more than 26 hours (e.g., lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or that involves expanding the cell population in vitro for, e.g., more than 3 days (e.g., expanding the cell population in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

In some embodiments, the cell population is enriched in IL6R expressing cells (e.g., cells that are positive for IL6Rα and/or IL6Rβ) prior to the start of the manufacturing method (e.g., prior to the start of the cytokine method or the activation method described herein). In some embodiments, the cell population comprises, e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% IL6R expressing cells (e.g., cells that are positive for IL6Rα and/or IL6Rβ) at the start of the manufacturing method (e.g., at the start of the cytokine method or the activation method described herein).

Method using cytokines

In some embodiments, the invention provides methods for producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR) (e.g., one or more CARs, such as two CARs), comprising (1) contacting the population of cells with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6, or a combination thereof, (2) contacting the population of cells (e.g., T cells) with a nucleic acid molecule(s) (e.g., a DNA or RNA molecule) encoding(s) the CAR, thereby producing a population of cells (e.g., T cells) comprising the nucleic acid molecule, and (3) harvesting the population of cells (e.g., T cells) for storage (e.g., reconstituting the population of cells in cryopreservation medium) or administration, wherein (a) step (2) is performed concurrently with step (1) or within 5 hours after the start of step (1), such as no later than 1, 2, 3, 4 or 5 hours after the start of step (1), and step (3) is performed no later than 26 hours after the start of step (1), such as no later than 22, 23 or 24 hours after the start of step (1), such as no later than 24 hours after the start of step (1), or (b) the cell population of step (3) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, such as no more than 10%, such as as assessed by the number of live cells, compared to the cell population at the start of step (1). In some embodiments, the nucleic acid molecule of step (2) is a DNA molecule. In some embodiments, the nucleic acid molecule of step (2) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (2) is in a viral vector, such as a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (2) is in a non-viral vector. In some embodiments, the nucleic acid molecule of step (2) is in a plasmid. In some embodiments, the nucleic acid molecule of step (2) is not in any vector. In some embodiments, step (2) involves transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule(s) encoding a CAR. In some embodiments, the cells are engineered to comprise a nucleic acid molecule encoding two CARs disclosed herein (e.g., a BCMA CAR and a CD19 CAR disclosed herein). In some embodiments, the cells are engineered to contain a nucleic acid molecule encoding a CAR in a diabody format disclosed herein, such as a CAR for BCMA/CD19 in a diabody format disclosed herein. In some embodiments, the cells are engineered to contain two nucleic acid molecules, each encoding a CAR disclosed herein (e.g., one nucleic acid molecule encoding a CAR for BCMA and one nucleic acid molecule encoding a CAR for CD19).

In some embodiments, a population of cells (e.g., T cells) is collected from a sample obtained by apheresis (e.g., a sample obtained by leukapheresis) from a subject.

In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed and T cells (e.g., CD4+ T cells and/or CD8+ T cells) are collected from the apheresis sample, such as using a cell sorting device (e.g., a CliniMACS® Prodigy® device). The collected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are then seeded to manufacture CAR-T using the cytokine-based method described herein. In some embodiments, at the end of the cytokine-based method, the CAR-T cells are cryopreserved and subsequently thawed and administered to the subject. In some embodiments, selected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are subjected to one or more freeze-thaw cycles prior to seeding to make CAR-T.

In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and transported as a fresh product (e.g., a non-frozen product) to a cell manufacturing facility. T cells (e.g., CD4+ T cells and/or CD8+ T cells) are isolated from the apheresis sample, such as by a cell sorting device (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are then seeded to manufacture CAR-T using the cytokine-based method described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are subjected to one or more freeze-thaw cycles prior to seeding to manufacture CAR-T.

In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. T cells (e.g., CD4+ T cells and/or CD8+ T cells) are collected from the apheresis sample, such as using a cell sorting device (e.g., a CliniMACS® Prodigy® device). The collected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are then transported as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The collected T cells (e.g., CD4+ T cells and/or CD8+ T cells) are subsequently thawed and seeded for CAR-T manufacturing using the cytokine-based method described herein.

In some embodiments, after seeding the cells (e.g., T cells), one or more cytokines (e.g., one or more cytokines selected from IL-2, IL-7, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-21, or IL-6 (e.g., IL-6/sIL-6R)) and a vector (e.g., a lentiviral vector) (e.g., one or more vectors) encoding a CAR (e.g., one or more CARs) are added to the cells. After incubation for 20-24 hours, the cells are washed and formulated for storage or administration.

Unlike traditional approaches to making CAR-T, the cytokine-based method provided herein does not involve CD3 and/or CD28 stimulation or ex vivo expansion of T cells. T cells that are exposed to anti-CD3 antibodies and anti-CD28 antibodies and that are expanded ex vivo tend to differentiate toward a central memory cell phenotype. Without wishing to be bound by any theory, it is believed that the cytokine-based method provided herein maintains or enhances the expression of the undifferentiated T cell phenotype during CAR-T manufacturing, while providing a CAR-T product that can persist longer after infusion into a subject.

In some embodiments, the population of cells is contacted with one or more cytokines (e.g., one or more cytokines selected from IL-2, IL-7, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)), IL-21, or IL-6 (e.g., IL-6/sIL-6Ra).

In some embodiments, the population of cells is contacted with IL-2. In some embodiments, the population of cells is contacted with IL-7. In some embodiments, the population of cells is contacted with IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-21. In some embodiments, the population of cells is contacted with IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, the population of cells is contacted with IL-2 and IL-7. In some embodiments, the population of cells is contacted with IL-2 and IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-2 and IL-21. In some embodiments, the population of cells is contacted with IL-2 and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, the population of cells is contacted with IL-7 and IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-7 and IL-21. In some embodiments, the population of cells is contacted with IL-7 and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, the population of cells is contacted with IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) and IL-21. In some embodiments, the population of cells is contacted with IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, the population of cells is contacted with IL-21 and IL-6 (e.g., IL-6/sIL-6Ra). In some embodiments, the population of cells is contacted with IL-7, IL-15 (e.g., hetIL-15 (IL15/sIL-15Ra)) and IL-21. In some embodiments, the population of cells is further contacted with an LSD1 inhibitor. In some embodiments, the population of cells is further contacted with a MALT1 inhibitor.

In some embodiments, the population of cells is contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 U/mL of IL-2. In some embodiments, the population of cells is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/ml of IL-7. In some embodiments, the population of cells is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/ml of IL-15.

In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs).

In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs simultaneously with contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 hours after the start of contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 5 hours after the start of contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 4 hours after the start of contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 3 hours after the start of contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 2 hours after the start of contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with a nucleic acid molecule encoding a CAR occurs no later than 1 hour after the start of contacting the population of cells with one or more cytokines described above.

In some embodiments, the cell population is collected for storage or administration.

In some embodiments, the population of cells is collected for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the initiation of contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is collected for storage or administration no later than 26 hours after the initiation of contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is collected for storage or administration no later than 25 hours after the initiation of contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is collected for storage or administration no later than 24 hours after the initiation of contacting the population of cells with one or more cytokines described above. In some embodiments, the cell population is collected for storage or administration no later than 23 hours after the cell population is first contacted with one or more cytokines described above. In some embodiments, the cell population is collected for storage or administration no later than 22 hours after the cell population is first contacted with one or more cytokines described above.

In some embodiments, the cell population is not expanded ex vivo.

In some embodiments, the cell population is expanded to an increase in number by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, for example, as measured by the number of live cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded to an increase in number by no more than 5%, for example, as measured by the number of live cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded by no more than 10% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded by no more than 15% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded by no more than 20% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded by no more than 25% increase in number, for example, as measured by the number of live cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded to increase in number by no more than 30%, for example, as measured by the number of living cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded to increase in number by no more than 35%, for example, as measured by the number of living cells, compared to the cell population before it was contacted with one or more cytokines described above. In some embodiments, the cell population is expanded to increase in number by no more than 40%, for example, as measured by the number of living cells, compared to the cell population before it was contacted with one or more cytokines described above.

In some embodiments, the cell population is expanded for no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, such as as assessed by the number of living cells, compared to the cell population before it was exposed to one or more cytokines described above.

In some embodiments, the cell population is not contacted in vitro with an agent that stimulates the CD3/TCR complex (e.g., an anti-CD3 antibody) and/or an agent that stimulates a costimulatory molecule on the cell surface (e.g., an anti-CD28 antibody), or, if contacted, the duration of the contacting step is less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours.

In some embodiments, the cell population is contacted in vitro with an agent that stimulates the CD3/TCR complex (e.g., an anti-CD3 antibody) and/or an agent that stimulates a costimulatory molecule on the cell surface (e.g., an anti-CD28 antibody) for 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.

In some embodiments, a population of cells produced by a cytokine-using method provided herein exhibits a higher percentage of naive cells among CAR-expressing cells (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60% higher) compared to cells produced by an otherwise similar method that further comprises contacting the population of cells with, e.g., an agent that binds a CD3/TCR complex (e.g., an anti-CD3 antibody) and/or an agent that binds a costimulatory molecule on the surface of cells (e.g., an anti-CD28 antibody).

In some embodiments, the method using cytokines herein is performed in cell media containing no more than 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8% serum. In some embodiments, the method using cytokines provided herein is performed in cell media containing an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

Additional illustrative manufacturing methods

In some embodiments, cells, such as T cells or NK cells, are activated, such as using anti-CD3/anti-CD28 antibody-coated Dynabeads®, contacted with one or more nucleic acid molecules encoding a CAR (e.g., one or more CARs), and then expanded in vitro for, such as, 7, 8, 9, 10, or 11 days. In some embodiments, cells, such as T cells or NK cells, are selected from a fresh or cryopreserved sample obtained by leukapheresis, such as by positive or negative selection. In some embodiments, the cells are contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the cells are contacted with a nucleic acid molecule encoding two CARs disclosed herein (e.g., a CAR for BCMA and a CAR for CD19). In some embodiments, the cells are contacted with two nucleic acid molecules, one expressing a first CAR (e.g., a CAR for BCMA) and the other expressing a second CAR (e.g., a CAR for CD19). In some embodiments, the cells are contacted with a nucleic acid molecule encoding a CAR in diabody format (e.g., a CAR for BCMA/CD19 in diabody format disclosed herein).

Elutriation

In some embodiments, the methods described herein include an elutriation method that removes unwanted cells, such as monocytes and blast cells, resulting in improved enrichment of desired immune effector cells suitable for CAR expression. In one embodiment, the elutriation method described herein is optimized for enrichment of desired immune effector cells suitable for CAR expression from a previously frozen sample, such as a thawed sample. In some embodiments, the elutriation method described herein provides cells with improved purity compared to cells collected according to elutriation protocols known in the art. In some embodiments, the elutriation method described herein includes using an optimized viscosity of the starting sample, such as a cell sample, such as a thawed cell sample, by dilution with certain isotonic solutions (e.g., PBS), and using an optimized combination of flow rates and collection volumes for each fraction collected by the elutriation device. Exemplary elutriation methods that can be used in the present invention are described on pages 48-51 of WO 2017/117112, which is incorporated herein by reference in its entirety.

Density gradient centrifugation

The manufacture of an adoptive cell therapy product requires the processing of the desired cells, such as immune effector cells, from the complex mixture of blood cells and blood elements present in the starting peripheral blood materials obtained by apheresis. Peripheral blood lymphocyte samples have been successfully isolated by density gradient centrifugation in Ficoll solution. However, Ficoll is not the preferred reagent for isolating cells for therapeutic use because Ficoll is not suitable for clinical use. In addition, Ficoll contains glycol, which has the potential to be toxic to cells. In addition, Ficoll density gradient centrifugation of thawed apheresis products after cryopreservation produces a suboptimal T cell product, such as described in the examples herein. For example, in cell preparations isolated by density gradient centrifugation in Ficoll solution, a loss of T cells was observed in the final product with a relative increase in non-T cell cells, particularly undesirable B cells, blast cells, and monocytes.

Without wishing to be bound by theory, it is believed that immune effector cells, such as T cells, dehydrate during cryopreservation, becoming denser than fresh cells. Without wishing to be bound by theory, it is also believed that immune effector cells, such as T cells, remain denser longer than other blood cells, and are thus more easily lost during Ficoll density gradient separation than other cells. Accordingly, without wishing to be bound by theory, it is believed that medium with a density greater than that of Ficoll will provide improved recovery of the desired immune effector cells compared to Ficoll or other media with a similar density to Ficoll, such as 1.077 g/mL.

In some embodiments, the density gradient centrifugation method described herein includes using a density gradient medium containing iodixanol. In some embodiments, the density gradient medium comprises about 60% iodixanol in water.

In some embodiments, the method of density gradient centrifugation described herein includes using a density gradient medium having a density greater than the density of Ficoll. In some embodiments, the method of density gradient centrifugation described herein includes using a density gradient medium having a density greater than 1.077 g/mL, such as greater than 1.077 g/mL, greater than 1.1 g/mL, greater than 1.15 g/mL, greater than 1.2 g/mL, greater than 1.25 g/mL, greater than 1.3 g/mL, greater than 1.31 g/mL. In some embodiments, the density gradient medium has a density of about 1.32 g/mL.

Further embodiments of density gradient centrifugation are described on pages 51-53 of WO 2017/117112, which is incorporated herein by reference in its entirety.

Enrichment through selection

Provided herein are methods for selecting specific cells to provide improved enrichment of desired immune effector cells suitable for CAR expression. In some embodiments, the selection comprises positive selection, such as selection for desired immune effector cells. In some embodiments, the selection comprises negative selection, such as selection for unwanted cells, such as removal of unwanted cells. In some embodiments, the positive or negative selection methods described herein are performed under flow conditions, such as with a flow device, such as the flow device described herein. Exemplary positive and negative selection methods are described on pages 53-57 of WO 2017/117112, which is incorporated herein by reference in its entirety. The selection methods can be carried out under flow conditions, such as using a flow device, also referred to as a cell processing system, to further enrich the cell preparation with the desired immune effector cells, such as T cells suitable for expressing CAR. Exemplary flow devices are described on pages 57-70 of WO 2017/117112, which is incorporated herein by reference in its entirety. Exemplary methods for separating cells and removing beads are described on pages 70-78 of WO 2017/117112, which is incorporated herein by reference in its entirety.

Selection procedures are not limited to those described on pages 57-70 of WO 2017/117112. Negative selection of T cells by removing unwanted cells using Miltenyi beads with CD19, CD14 and CD26 antibodies in combination with column technology (CliniMACS® Plus or CliniMACS® Prodigy®) or positive selection of T cells using a combination of Miltenyi beads with CD4 and CD8 antibodies and column technology (CliniMACS® Plus or CliniMACS® Prodigy®) can be used. Alternatively, column-free technology with beads with releasable CD3 antibodies (GE Healthcare) can be used.

Additionally, pellet-free technologies such as the ThermoGenesis X-Series devices can also be used.

Routes of clinical application

All methods in this document can be performed according to clinical good manufacturing practice (cGMP) standards.

The methods can be used for purifying, enriching, collecting, washing, concentrating cells or for replacing media for cells, in particular during the collection of raw starting materials (in particular cells) at the beginning of the manufacturing method, as well as during the manufacturing method for selecting or expanding cells for cell therapy.

The cells may include any plurality of cells. The cells may be of the same cell type or a mixture of cell types. Additionally, the cells may be obtained from a single donor, such as an autologous donor or a single allogeneic donor, for cell therapy. The cells may be obtained from patients by, for example, leukapheresis or apheresis. The cells may include T cells, for example, may include a population that is greater than 50% T cells, greater than 60% T cells, greater than 70% T cells, greater than 80% T cells, or 90% T cells.

Selection methods may be particularly useful for selecting cells prior to culture and expansion. For example, paramagnetic particles coated with antibodies to CD3 and/or CD28 may be used to select T cells for expansion or to introduce a nucleic acid encoding a chimeric antigen receptor (CAR) or other protein. Such a method is used to generate CTL019 T cells for the treatment of acute lymphoblastic leukemia (ALL).

The bead removal methods and modules disclosed herein may be particularly useful in the production of cells for cell therapy, such as in the purification of cells before or after culture and expansion. For example, paramagnetic particles coated with anti-CD3 and/or anti-CD28 antibodies may be used to selectively expand T cells, such as those T cells that are or will be modified by the introduction of a nucleic acid encoding a chimeric antigen receptor (CAR) or other protein such that the CAR is expressed by the T cells. During the production of such T cells, bead removal methods or modules may be used to separate the T cells from the paramagnetic particles. Such a bead removal method or module is used to produce, for example, CTL019 T cells for the treatment of acute lymphoblastic leukemia (ALL).

In one such method, illustrated by way of example herein, cells, such as T cells, are collected from a donor (e.g., a patient to be treated with an autologous chimeric antigen receptor T cell product) by apheresis (e.g., leukapheresis). The collected cells may then optionally be purified, such as by an elutriation step or by positive or negative selection of target cells (e.g., T cells). Paramagnetic particles, such as anti-CD3/anti-CD28 coated paramagnetic particles, may then be added to the cell population to expand the T cells. The method may also include a transduction step wherein a nucleic acid encoding one or more desired proteins, such as a CAR, such as a CAR that targets CD19, is introduced into the cell. The nucleic acid may be introduced into a lentiviral vector. The cells, such as cells transduced with a lentiviral vector, can then be expanded over a period of several days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days, such as in the presence of a suitable medium. Following expansion, the bead removal methods/modules disclosed herein can be used to separate the desired T cells from the paramagnetic particles. The method can include one or more bead removal steps according to the methods of the present invention. The bead-removed cells can then be formulated for administration to a patient. Examples of CAR-T cells and their manufacture are further described, such as WO2012/079000, which is incorporated herein by reference in its entirety. The systems and methods of the present invention can be used for any of the cell separation/cell purification/bead removal methods from cells described in WO2012/079000 or related applications. Additional methods for making CAR-T are described, for example, in WO2016109410 and WO2017117112, which are incorporated herein by reference in their entireties.

The systems and methods described herein may similarly provide a beneficial effect for other cell therapy products by providing a reduction in the loss of desired cells, a reduction in the extent of cell damage, and a more reliable removal of magnetic and any non-paramagnetic particles from cells with less or no chemical exposure compared to traditional systems and methods.

Although only illustrative embodiments of the present invention have been specifically described above, it will be understood that modifications and variations of such examples are possible without departing from the spirit and intended scope of the present invention. For example, magnetic modules and systems containing them can be arranged and used in a variety of configurations in addition to those described. In addition, non-magnetic modules can also be used. In addition, systems and methods can include additional components and steps that are not specifically described herein. For example, the methods can include a primary treatment where a liquid is first introduced into a component to remove bubbles and reduce resistance to movement of the cell suspension or buffer. In addition, embodiments can include only a portion of the systems described herein for use with the methods described herein. For example, embodiments can relate to disposable modules, sleeves, etc., usable within non-disposable equipment to form an integral system capable of separating cells or removing beads from cells to produce a cell-based product.

Additional manufacturing methods and processes that can be used in combination with the present invention have been described in the prior art. For example, improved washing steps and an improved manufacturing method are described on pages 86-91 of WO 2017/117112.

Sources of immune effector cells

This section provides additional methods or steps for obtaining a source sample containing the desired immune effector cells, isolating and processing the desired immune effector cells, such as T cells, and removing unwanted materials, such as unwanted cells. The additional methods or steps described in this section can be used in combination with any of the elutriation, density gradient centrifugation, flow selection, or enhanced washing step described in the previous sections.

A source of cells, such as T cells or natural killer (NK) cells, may be obtained from a subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic forms thereof. T cells may be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

In some embodiments of the present invention, immune effector cells, such as T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art and any of the methods disclosed herein, in any combination of steps thereof. In some embodiments, cells are obtained from circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and optionally place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution is free of calcium and may be free of magnesium or may be free of many, if not all, divalent cations. In some embodiments, the cells are washed using the improved washing step described herein.

The initial steps of activation in the absence of calcium may result in enhanced activation. As will be apparent to those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art, such as using a semiautomated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, CytoMate™ from Baxter, or Cell Saver 5 from Haemonetics), Cell Saver Elite from Haemonetics (Sepax or Sefia from GE Healthcare), or a device using spin membrane filtration technology (LOVO from Fresenius Kabi), according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free and Mg-free PBS, PlasmaLyte A, PBS-EDTA supplemented with human serum albumin (HSA), or other buffered or unbuffered saline. Alternatively, unwanted components from the apheresis sample can be removed and the cells can be resuspended directly in the culture medium.

In some embodiments, the desired immune effector cells, such as T cells, are isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, such as by PERCOLL™ gradient centrifugation or by countercurrent elutriation centrifugation.

The methods described herein may include, for example, selecting a specific subpopulation of immune effector cells, for example, T cells, that is a population of cells depleted of regulatory T cells, for example, depleted of CD25+ cells or depleted of ^CD25high cells, using, for example, a negative selection technique, for example, as described herein. In some embodiments, the population of cells depleted of regulatory T cells comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells or ^CD25high cells.

In some embodiments, regulatory T cells, such as CD25+ T cells or CD25 ^high T cells, are removed from the population using an anti-CD25 antibody or fragment thereof or a CD25-binding ligand, such as IL-2. In some embodiments, the anti-CD25 antibody or fragment thereof or the CD25-binding ligand is conjugated to a substrate, such as a bead, or otherwise coated with a substrate, such as a bead. In some embodiments, the anti-CD25 antibody or fragment thereof is conjugated to a substrate as described herein.

In some embodiments, regulatory T cells, such as CD25+ T cells or CD25 ^high T cells, are removed from the population using Miltenyi™ CD25 Depletion Reagent. In some embodiments, the ratio of cells to CD25 Depletion Reagent is 1e7 cells per 20 μl, or 1e7 cells per 15 μl, or 1e7 cells per 10 μl, or 1e7 cells per 5 μl, or 1e7 cells per 2.5 μl, or 1e7 cells per 1.25 μl. In some embodiments, for example, for regulatory T cells, greater than 500 million cells/mL are used. In some embodiments, a cell concentration of 600, 700, 800, or 900 million cells/mL is used.

In some embodiments, the immune effector cell population to be depleted comprises about 6× ¹⁰⁹ CD25+ T cells. In some embodiments, the immune effector cell population to be depleted comprises from about 1× ¹⁰⁹ to 1× ¹⁰¹⁰ CD25+ T cells, including any integer value therebetween. In some embodiments, the resulting T regulatory cell depleted population comprises 2× ¹⁰⁹ regulatory T cells, such as CD25+ cells or ^CD25high cells, or less (e.g., 1× ¹⁰⁹ , 5× ¹⁰⁸ , 1× ¹⁰⁸ , 5× ¹⁰⁷ , 1× ¹⁰⁷ or less regulatory T cells).

In some embodiments, regulatory T cells, such as CD25+ cells or CD25 ^high cells, are removed from the population using the CliniMACS system with a set of depletion tubes, such as, for example, 162-01 tubes. In some embodiments, the CliniMACS system operates with established depletion parameters, such as, for example, DEPLETION2.1.

Without being limited by a particular theory, it is believed that by reducing the level of negative regulators of immune cells (e.g., reducing the number of unwanted immune cells, such as Treg cells) in a subject prior to apheresis or during the manufacture of a CAR-expressing cell product, the risk of relapse in the subject is significantly reduced. For example, methods of depleting Treg cells are known in the art. Methods of depleting Treg cells include, but are not limited to, cyclophosphamide, an anti-GITR antibody (the anti-GITR antibody described herein), a CD25 depletion reagent, and combinations thereof.

In some embodiments, the manufacturing methods comprise reducing the number (e.g., depleting the population) of Treg cells prior to manufacturing the CAR-expressing cell. For example, the manufacturing methods comprise contacting a sample, e.g., a sample obtained by apheresis, with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment thereof or CD25-binding ligand), e.g., to deplete Treg cells prior to manufacturing the CAR-expressing cell product (e.g., T cells, NK cells).

Without being limited by a particular theory, it is believed that by reducing the level of negative regulators of immune cells (e.g., reducing the number of unwanted immune cells, such as Treg cells) in a subject prior to apheresis or during the manufacture of a CAR-expressing cell product, the risk of relapse in the subject may be reduced. In some embodiments, the subject is pretreated with one or more therapies that reduce the number of Treg cells prior to harvesting the cells for the manufacture of a CAR-expressing cell product, thereby reducing the risk of relapse in the subject when treated with CAR-expressing cells. In some embodiments, methods of reducing the number of Treg cells include, but are not limited to, administering to the subject one or more of cyclophosphamide, an anti-GITR antibody, a CD25 depletion reagent, or a combination thereof. In some embodiments, methods of reducing the number of Treg cells include, but are not limited to, administering to the subject one or more of cyclophosphamide, an anti-GITR antibody, a CD25 depletion reagent, or a combination thereof. Administration of one or more of cyclophosphamide, an anti-GITR antibody, a CD25 depletion reagent, or a combination thereof may occur before, during, or after infusion of the CAR-expressing cell-based product. Administration of one or more of cyclophosphamide, an anti-GITR antibody, a CD25 depletion reagent, or a combination thereof may occur before, during, or after infusion of the CAR-expressing cell-based product.

In some embodiments, the manufacturing methods comprise reducing the number (e.g., depleting the population) of Treg cells prior to manufacturing the CAR-expressing cell. For example, the manufacturing methods comprise contacting a sample, e.g., a sample obtained by apheresis, with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment thereof or CD25-binding ligand), e.g., to deplete Treg cells prior to manufacturing the CAR-expressing cell product (e.g., T cells, NK cells).

In some embodiments, the subject is pretreated with cyclophosphamide prior to harvesting the cells for the manufacture of a CAR-expressing cell product, thereby reducing the risk of relapse in the subject when treated with CAR-expressing cells (e.g., treatment with CTL019). In some embodiments, the subject is pretreated with an anti-GITR antibody prior to harvesting the cells for the manufacture of a CAR-expressing cell product (e.g., T cells or NK cells), thereby reducing the risk of relapse in the subject when treated with CAR-expressing cells.

In some embodiments, a method for making CAR-expressing cells (e.g., T cells, NK cells) is modified to provide for depletion of Treg cells prior to making a CAR-expressing cell product (e.g., T cells, NK cells) (e.g., a CTL019 product). In some embodiments, CD25 depletion is used to deplete Treg cells prior to making a CAR-expressing cell product (e.g., T cells or NK cells) (e.g., a CTL019 product).

In some embodiments, the cell population to be removed is neither regulatory T cells nor tumor cells, but is cells that otherwise negatively affect the expansion and/or function of CAR T cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potential immunosuppressive cells. In some embodiments, it is contemplated that such cells are removed simultaneously with the regulatory T cells and/or tumor cells, or after said depletion, or in another order.

The methods described herein may include more than one selection step, such as more than one depletion step. Enrichment of a T cell population by negative selection may be accomplished, for example, by using a combination of antibodies directed to surface markers unique to cells subject to negative selection. One method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry, which utilizes a cocktail of monoclonal antibodies directed to cell surface markers present on cells subject to negative selection. For example, to enrich CD4+ cells by negative selection, a cocktail of monoclonal antibodies may comprise antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein may further comprise removing from the population cells expressing a tumor antigen, e.g., a tumor antigen that does not include CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby providing a population depleted of regulatory T cells, e.g., depleted of CD25+ cells or depleted of ^CD25high cells and depleted of cells with a tumor antigen, that is suitable for expressing a CAR, e.g., a CAR described herein. In some embodiments, cells expressing a tumor antigen are removed simultaneously with regulatory T cells, e.g., CD25+ cells or ^CD25high cells. For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof may be attached to the same substrate, such as a bead, that can be used to remove cells, or an anti-CD25 antibody or fragment thereof or an anti-tumor antigen antibody or fragment thereof may be attached to separate beads, a mixture of which can be used to remove cells. In other embodiments, the removal of regulatory T cells, such as CD25+ cells or CD25 ^high cells, and the removal of cells expressing a tumor antigen are sequential and may occur, for example, in any order.

Also provided are methods that include removing from a population of cells expressing an immune checkpoint inhibitor, e.g., an immune checkpoint inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, thereby providing a population depleted of regulatory T cells, e.g., depleted of CD25+ cells, and depleted of cells with an immune checkpoint inhibitor, e.g., depleted of PD1+, LAG3+, and/or TIM3+ cells. Exemplary immune checkpoint inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecule, MHC class II molecule, GAL9, adenosine, and TGF (e.g., TGF-beta), such as described herein. In some embodiments, cells expressing an immune checkpoint inhibitor are co-depleted with regulatory T cells, such as CD25+ cells or ^CD25high cells. For example, an anti-CD25 antibody or fragment thereof and an anti-immune checkpoint inhibitor antibody or fragment thereof may be attached to the same bead that can be used to remove cells, or an anti-CD25 antibody or fragment thereof and an anti-immune checkpoint inhibitor antibody or fragment thereof may be attached to separate beads, a mixture of which can be used to remove cells. In other embodiments, the removal of regulatory T cells, such as CD25+ cells or CD25 ^high cells, and the removal of cells expressing an immune checkpoint inhibitor are sequential and may occur, for example, in any order.

The methods described herein may include a positive selection step. For example, T cells can be isolated by incubation with beads conjugated to anti-CD3 antibodies/anti-CD28 antibodies (e.g., 3x28), such as Dynabeads® M-450 CD3/CD28 T, for a period of time sufficient to positively select the desired T cells. In some embodiments, the period of time is about 30 minutes. In some embodiments, the period of time is between 30 minutes and 36 hours or more, and includes all integer values therebetween. In some embodiments, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period of time is between 10 and 24 hours, such as 24 hours. Longer incubation times can be used to isolate T cells in any situation in which T cells are few in number compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, using longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time that T cells are able to bind to the anti-CD3/CD28 antibody beads and/or increasing or decreasing the ratio of beads to T cells (as further described herein), positive or negative selection of T cell subsets can be preferentially achieved at the start of the culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD3 antibodies and/or anti-CD28 antibodies on the beads or other surface, positive or negative selection of T cell subsets can be preferentially achieved at the start of the culture or at other time points as desired.

In some embodiments, a population of T cells that express one or more of IFNγ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin or other relevant molecules, such as other cytokines, can be selected. Screening methods for cellular expression can be determined, for example, by the methods described in PCT Publication No. WO 2013/126712.

To isolate a desired cell population by positive or negative selection, the cell concentration and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be necessary to significantly reduce the volume in which the beads and cells are mixed with each other (e.g., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, in some embodiments, a concentration of 10 billion cells/mL, 9 billion/mL, 8 billion/mL, 7 billion/mL, 6 billion/mL, or 5 billion/mL is used. In some embodiments, a concentration of 1 billion cells/mL is used. In some embodiments, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In some embodiments, concentrations of 125 or 150 million cells/mL can be used.

The use of high concentrations may result in increased cell yield, cell activation, and cell proliferation. In addition, the use of high cell concentrations may allow more efficient capture of cells that may have weak expression of the target antigens of interest, such as CD28-negative T cells, or cells from samples that contain many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and may need to be recovered. For example, the use of high cell concentrations may allow more efficient selection of CD8+ T cells, which typically have weaker CD28 expression.

In some embodiments, it may be necessary to use lower concentrations of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express the desired antigens to be bound to the particles in high quantities. For example, CD4+ T cells express CD28 at higher levels and are captured at low concentrations more efficiently than CD8+ T cells. In some embodiments, the concentration of cells used is 5×10 ⁶ /mL. In some embodiments, the concentration used may be from about 1×10 ⁵ /mL to 1×10 ⁶ /mL, including any integer value therebetween.

In some embodiments, cells may be incubated on a rotator for varying periods of time at varying speeds at 2-10°C or at room temperature.

In some embodiments, the plurality of immune effector cells in the population do not express diacylglycerol kinase (DGK), such as being deficient in DGK. In some embodiments, the plurality of immune effector cells in the population do not express Ikaros, such as being deficient in Ikaros. In some embodiments, the plurality of immune effector cells in the population do not express both DGK and Ikaros, such as being deficient in both DGK and Ikaros.

T cells for stimulation may also be frozen after a wash step. Without being bound by theory, it is believed that the freeze and thaw step provides a more homogeneous product by removing granulocytes and to some extent monocytes from the cell population. Following a wash step that removes plasma and platelets, the cells may be suspended in a freezing solution. Although many freezing solutions and freezing parameters are known in the art and will be applicable herein, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% dextran 40 in 5% dextrose, 20% human serum albumin and 7.5% DMSO or 31.25% PlasmaLyte-A, 31.25% solution with 5% dextrose in 0.45% NaCl, 10% dextran 40 in 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1° per minute and storing in the vapor phase at liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

In some embodiments, cryopreserved cells are thawed and washed as described herein and left for one hour at room temperature prior to activation using the methods of the present invention.

The present invention also provides for the collection of a blood sample or apheresis product from a subject at a time before the expanded cells described herein may be needed. As such, a source of cells to be expanded may be collected at any desired time, and the desired cells, such as T cells, may be isolated and frozen for subsequent use in immune effector cell therapy for any disease or condition that would benefit from immune effector cell therapy, such as those described herein. In some embodiments, the blood sample or apheresis sample is collected from a generally healthy subject. In some embodiments, the blood sample or apheresis sample is collected from a generally healthy subject who is at risk for developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for subsequent use. In some embodiments, the T cells may be expanded, frozen, and used at a later date. In some embodiments, samples are collected from a patient shortly after diagnosis of a particular disease described herein, but prior to any treatments. In some embodiments, cells are isolated from a blood sample or apheresis product from a subject prior to any number of appropriate treatments, including but not limited to treatments such as natalizumab, efalizumab, antivirals, chemotherapy, radiation therapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunodestructive agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

In some embodiments of the present invention, T cells are obtained from a patient immediately following a treatment whereby the subject retains functional T cells. In this regard, it has been observed that following certain cancer treatments, particularly treatments with drugs that damage the immune system, shortly after treatment during the period when patients are typically recovering from treatment, the quality of the T cells obtained may be optimal or improved in terms of their ability to proliferate ex vivo. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a state favorable for improved engraftment and expansion in vivo. Thus, in the context of the present invention, collecting blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, during this recovery phase is contemplated. In addition, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject that is favorable for repopulation, recycling, regeneration, and/or expansion of certain cell types, especially during a certain time window after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

In some embodiments, immune effector cells expressing a CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject that has received a low dose, immune response enhancing mTOR inhibitor. In some embodiments, a population of immune effector cells, e.g., T cells, engineered to express a CAR are collected after a sufficient amount of time or after sufficient administration of a low dose, immune response enhancing mTOR inhibitor, such that the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells, to PD1-positive immune effector cells, e.g., T cells, in the subject or in material collected from the subject is at least transiently increased.

In other embodiments, a population of immune effector cells, such as T cells, that have been or will be engineered to express a CAR can be treated ex vivo by contacting it with an amount of an mTOR inhibitor that provides an increase in the number of PD1-negative immune effector cells, such as T cells, or provides an increase in the ratio of PD1-negative immune effector cells, such as T cells, to PD1-positive immune effector cells, such as T cells.

It is generally accepted that the methods of use may employ culture media conditions comprising human AB serum at a concentration of 5% or less, such as 2%, and employ known culture media conditions and compositions, such as those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS™ Immune Cell Serum Replacement" Clinical & Translational Immunology (2015) 4, e31; doi:10.1038/cti.2014.31.

In some embodiments, the methods of use may utilize medium conditions comprising at least about 0.1%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, or 10% serum. In some embodiments, the medium comprises about 0.5%-5%, about 0.5%-4.5%, about 0.5%-4%, about 0.5%-3.5%, about 0.5%-3%, about 0.5%-2.5%, about 0.5%-2%, about 0.5%-1.5%, about 0.5%-1.0%, about 1.0%-5%, about 1.5%-5%, about 2%-5%, about 2.5%-5%, about 3%-5%, about 3.5%-5%, about 4%-5%, or about 4.5%-5% serum. In some embodiments, the medium comprises about 0.5% serum. In some embodiments, the medium comprises about 0.5% serum. In some embodiments, the medium comprises about 1% serum. In some embodiments, the medium comprises about 1.5% serum. In some embodiments, the medium comprises about 2% serum. In some embodiments, the medium comprises about 2.5% serum. In some embodiments, the medium comprises about 3% serum. In some embodiments, the medium comprises about 3.5% serum. In some embodiments, the medium comprises about 4% serum. In some embodiments, the medium comprises about 4.5% serum. In some embodiments, the medium comprises about 5% serum. In some embodiments, the serum is human serum, such as human AB serum. In some embodiments, the serum is human serum that has been allowed to clot naturally after collection, such as over-the-counter (OTC) serum. In some embodiments, the serum is human plasma-derived serum. Plasma-derived serum can be obtained by removing fibrin from pooled human plasma collected in the presence of an anticoagulant, such as sodium citrate.

In some embodiments, the methods of use may utilize culture media conditions that include serum-free medium. In some embodiments, the serum-free medium is OpTmizer™ CTS™ (LifeTech), ImmunoCult™ XF (Stemcell Technologies), CellGro™ (CellGenix), TexMacs™ (Miltenyi), Stemline™ (Sigma), Xvivo15™ (Lonza), PrimeXV® (Irvine Scientific), or StemXVivo® (R&D Systems). The serum-free medium may be supplemented with a serum simulant, such as ICSR (Immune Cell Serum Replacer) from LifeTech. The level of serum simulant (e.g., ICSR) may be, for example, up to 5%, such as about 1%, 2%, 3%, 4%, or 5%. In some embodiments, the serum-free medium can be supplemented with serum, such as human serum, such as human AB serum. In some embodiments, the serum is human serum that has been allowed to clot naturally after collection, such as over-the-counter (OTC) serum. In some embodiments, the serum is human plasma-derived serum. Plasma-derived serum can be obtained by removing fibrin from pooled human plasma collected in the presence of an anticoagulant, such as sodium citrate.

In some embodiments, the T cells in the population are deficient in diacylglycerol kinase (DGK). DGK-deficient cells include cells that do not express DGK as RNA or protein or have reduced or inhibited DGK activity. DGK-deficient cells can be generated using genetic approaches, such as by administering RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with the DGK inhibitors described herein.

In some embodiments, the T cells in the population are deficient in Ikaros. Ikaros-deficient cells include cells that do not express Ikaros as RNA or protein or have reduced or inhibited Ikaros activity; Ikaros-deficient cells can be produced by genetic approaches, such as by introducing RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be produced by treatment with Ikaros inhibitors, such as lenalidomide.

In some embodiments, the T cell population is DGK deficient and Ikaros deficient, such as lacking DGK and Ikaros expression or having reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros deficient cells can be obtained by any of the methods described herein.

In some embodiments, the NK cells are obtained from a subject. In some embodiments, the NK cells are an NK cell line, such as an NK-92 cell line (Conkwest).

Allogeneic CAR expressing cells

In some embodiments described herein, the immune effector cell may be an allogeneic immune effector cell, such as a T cell or an NK cell. For example, the cell may be an allogeneic T cell, such as an allogeneic T cell that lacks expression of a functional T cell receptor (TCR) and/or a human leukocyte antigen (HLA), such as HLA class I and/or HLA class II.

A T cell that lacks a functional TCR may, for example, be engineered not to express a functional TCR on its surface at all, engineered not to express one or more of the subunits that form a functional TCR (e.g., engineered not to express (or to exhibit reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and/or TCR zeta), or engineered to produce very little functional TCR on its surface. Alternatively, the T cell may express a substantially impaired TCR, such as by expressing mutant or truncated forms of one or more TCR subunits. The term "substantially impaired TCR" means that the TCR will not elicit an unwanted immune response in the host.

A T cell as described herein can, for example, be engineered to not express functional HLA on its surface. For example, a T cell as described herein can be engineered to suppress expression of HLA, such as HLA class I and/or HLA class II, on the cell surface. In some embodiments, suppression of HLA expression can be accomplished by reducing or eliminating expression of beta-2 microglobulin (B2M).

In some embodiments, the T cell may lack a functional TCR and a functional HLA, such as HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA can be produced by any suitable method, including by knocking out or knocking down one or more subunits of the TCR or HLA. For example, knocking down of the TCR and/or HLA in a T cell can be envisaged using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

In some embodiments, the allogeneic cell may be a cell that does not express or expresses at low levels an inhibitory molecule, such as by any method described herein. For example, the cell may be a cell that does not express or expresses at low levels an inhibitory molecule, such as a molecule that can reduce the ability of a cell expressing a CAR to carry out an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecule, MHC class II molecule, GAL9, adenosine, and TGF (e.g., TGF-beta). Inhibition of an inhibitory molecule, such as by inhibition at the DNA, RNA, or protein level, can optimize the functional characteristics of a cell expressing a CAR. In some embodiments, an inhibitory nucleic acid may be used, such as an inhibitory nucleic acid such as a dsRNA, such as an siRNA or shRNA, a clustered regularly interspaced short palindromic repeat (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), such as those described herein.

siRNA and shRNA for TCR or HLA inhibition

In some embodiments, TCR expression and/or HLA expression can be inhibited by siRNA or shRNA targeting a nucleic acid encoding a TCR and/or HLA and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecule, MHC class II molecule, GAL9, adenosine, and TGF-beta) in a cell, such as a T cell.

Expression systems for siRNA and shRNA and exemplary shRNAs are described, for example, in paragraphs 649 and 650 of international application WO2015/142675, filed March 13, 2015, which is incorporated by reference in its entirety.

CRISPR for TCR or HLA inhibition

As used herein, "CRISPR" or "CRISPR for TCR and/or HLA" or "CRISPR for inhibition of TCR and/or HLA" refers to a clustered regularly interspaced short palindromic repeat array or a system containing such a repeat array. As used herein, "Cas" refers to CRISPR-associated protein. The "CRISPR/Cas system" refers to a system derived from CRISPR and Cas that can be used to silence or mutate a TCR and/or HLA gene and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecules, MHC class II molecules, GAL9, adenosine, and TGF-beta) in a cell, such as a T cell.

The CRISPR/Cas system and its uses are described, for example, in paragraphs 651-658 of international application WO2015/142675, filed March 13, 2015, which is incorporated by reference in its entirety.

TALENs for TCR and/or HLA inhibition

"TALEN" or "TALEN for HLA and/or TCR" or "TALEN for inhibiting HLA and/or TCR" refers to a transcription activator-like effector nuclease, which is an artificial nuclease that can be used to edit an HLA and/or TCR gene and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecules, MHC class II molecules, GAL9, adenosine and TGF-beta) in a cell, such as a T cell.

TALENs and their uses are described, for example, in paragraphs 659-665 of international application WO2015/142675, filed March 13, 2015, which is incorporated by reference in its entirety.

Zinc finger nuclease for inhibition of HLA and/or TCR

"ZFN" or "zinc finger nuclease" or "ZFN for HLA and/or TCR" or "ZFN for inhibiting HLA and/or TCR" refers to a zinc finger nuclease, which is an artificial nuclease that can be used to edit an HLA and/or TCR gene and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I molecules, MHC class II, GAL9, adenosine, and TGF-beta) in a cell, such as a T cell.

ZFNs and their uses are described, for example, in paragraphs 666-671 of international application WO2015/142675, filed March 13, 2015, which is incorporated by reference in its entirety.

Telomerase expression

Telomeres play a critical role in the persistence of somatic cells and their length is maintained by telomerase (TERT). Telomere length can be very short in CLL cells (Roth et al., "Significantly shorter telomeres in T-cells of patients with ZAP-70+/CD38 chronic lymphocytic leukaemia" British Journal of Haematology, 143, 383-386., August 28 2008) and can be even shorter in engineered CAR-expressing cells, such as CART19 cells, limiting their expansion potential after adoptive transfer into a patient. Telomerase expression can "rescue" CAR-expressing cells from replicative exhaustion.

Without wishing to be bound by any particular theory, it is believed that in some embodiments, a therapeutic T cell has short-term persistence in a patient due to shortened telomeres in the T cell; accordingly, transfection with a telomerase gene may result in lengthening of telomeres in the T cell and improved persistence of the T cell in the patient. See Carl June, "Adoptive T cell therapy for cancer in the clinic," Journal of Clinical Investigation, 117:1466-1476 (2007). Thus, in some embodiments, an immune effector cell, e.g., a T cell, has ectopic expression of a telomerase subunit, e.g., a telomerase catalytic subunit, e.g., TERT, e.g., hTERT. In some embodiments, the present invention provides a method of producing a cell expressing a CAR, comprising contacting the cell with a nucleic acid encoding a telomerase subunit, e.g., a telomerase catalytic subunit, e.g., TERT, e.g., hTERT. The cell may be contacted with the nucleic acid before, simultaneously with, or after contacting the construct encoding the CAR.

Telomerase expression may be stable (e.g., the nucleic acid may integrate into the cell genome) or transient (e.g., the nucleic acid does not integrate and the expression level decreases after some period of time, such as a few days). Stable expression may be achieved by transfecting or transducing a cell with DNA encoding a telomerase subunit and a selectable marker and selecting for stable integrants. Alternatively or in combination, stable expression may be achieved by site-specific recombination, such as with the Cre/Lox or FLP/FRT system.

Transient expression may involve transfection or transduction with a nucleic acid, such as DNA or RNA, such as mRNA. In some embodiments, transient transfection of mRNA avoids the genetic instability sometimes associated with stable transfection of TERT. Transient expression of exogenous telomerase activity is described, for example, in International Application WO2014/130909, which is incorporated herein by reference in its entirety. In some embodiments, transfection of the telomerase subunit using mRNA is performed in accordance with the messenger RNA Therapeutics™ platform commercialized by Moderna Therapeutics. For example, the method may be the method described in U.S. Patent Nos. 8,710,200, 8,822,663, 8,680,069, 8,754,062, 8,664,194, or 8,680,069.

In some embodiments, hTERT has the amino acid sequence of GenBank protein ID AAC51724.1 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization," Cell, volume 90, issue 4, August 22, 1997, pages 785-795): MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLL RRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEAT SLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAG VCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTET TFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELY FVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDM ENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQ VNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD (SEQ ID NO: 284)

In some embodiments, hTERT has a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 284. In some embodiments, hTERT has the sequence of SEQ ID NO: 284. In some embodiments, hTERT comprises a deletion (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both. In some embodiments, hTERT comprises a transgenic amino acid sequence (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both.

In some embodiments, hTERT is encoded by the nucleic acid sequence of GenBank Accession No. AF018167 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization," Cell, volume 90, issue 4, August 22, 1997, pages 785-795).

Activation and expansion of immune effector cells (eg, T cells)

Immune effector cells, such as T cells, obtained or enriched by the methods described herein can be activated and expanded, typically by the methods described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005.

Typically, a population of immune effector cells can be expanded by contacting them with a surface to which are attached an agent that stimulates signaling associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of T cells. In particular, the populations of T cells can be stimulated as described herein, such as by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. To costimulate an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of the anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and can be used as well as other methods generally known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In some embodiments, the primary stimulatory signal and the costimulatory signal for a T cell can be provided using different protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" arrangement) or to separate surfaces (i.e., in a "trans" arrangement). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In some embodiments, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In some embodiments, both agents can be in solution. In some embodiments, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents. In this regard, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 regarding artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

In some embodiments, the two agents are immobilized on the beads, either on the same bead, i.e., in the "cis" position, or on separate beads, i.e., in the "trans" position. As an example, the agent that provides the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent that provides the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized on the same bead in equivalent molecular amounts. In some embodiments, each antibody bound to the beads is used in a 1:1 ratio to expand CD4+ T cells and grow T cells. In some embodiments, the ratio of anti-CD3:CD28 antibodies bound to the beads is such that an increase in the rate of T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In some embodiments, there is an increase of about 1 to about 3 times compared to the expansion observed using a 1:1 ratio. In some embodiments, the ratio of anti-CD3:CD28 antibodies bound to the beads is in the range of 100:1 to 1:100 and includes all integer values therebetween. In some embodiments, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 antibodies is less than one. In some embodiments, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In some embodiments, the anti-CD3 and CD28 antibodies bound to the beads are used in a ratio of 1:100. In some embodiments, the anti-CD3 and CD28 antibodies bound to the beads are used in a ratio of 1:75. In some embodiments, the anti-CD3 and anti-CD28 antibodies bound to the beads are used in a ratio of 1:50. In some embodiments, the anti-CD3 and anti-CD28 antibodies bound to the beads are used in a ratio of 1:30. In some embodiments, the anti-CD3 and anti-CD28 antibodies bound to the beads are used in a ratio of 1:10. In some embodiments, the anti-CD3 and anti-CD28 antibodies bound to the beads are used in a ratio of 1:3. In some embodiments, the anti-CD3 and anti-CD28 antibodies bound to the beads are used in a ratio of 3:1.

For stimulation of T cells or other target cells, particle to cell ratios of 1:500 to 500:1, including any integer values in this range, can be used. As will be apparent to those of ordinary skill in the art, the particle to cell ratio can depend on the size of the particles relative to the target cell. For example, small beads can bind only a few cells, while larger beads can bind many cells. In some embodiments, the cell to particle ratio is in the range of 1:100 to 100:1 and includes any integer values in this range, and in some embodiments, the ratio is from 1:9 to 9:1 and includes any integer values in this range, and can also be used to stimulate T cells. The ratio of particles bound to anti-CD3 antibodies and anti-CD28 antibodies to T cells that results in stimulation of T cells may vary as noted above, but certain suitable values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one suitable ratio being at least 1:1 for particles per T cell. In some embodiments, a particle to cell ratio of 1:1 or less is used. In some embodiments, a suitable particle:cell ratio is 1:5. In some embodiments, the particle to cell ratio can be varied depending on the day of stimulation. For example, in some embodiments, the particle to cell ratio is from 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day thereafter for up to 10 days at final ratios of 1:1 to 1:10 (based on the number of cells on the day of addition). In some embodiments, the particle to cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on the third and fifth days of stimulation. In some embodiments, the particles are added every day or every other day to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In some embodiments, the particle to cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In some embodiments, the particles are added every day or every other day to a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. One skilled in the art will understand that many other ratios may be suitable for use in the present invention. In particular, the ratios will vary depending on the size of the particles, as well as the size and type of cells. In some embodiments, the most typical ratios for use are about 1:1, 2:1 and 3:1 on the first day.

In some embodiments, cells such as T cells are combined with beads coated with an agent, the beads and cells are subsequently separated, and the cells are then cultured. In some embodiments, the beads coated with an agent and the cells are not separated before culturing, but are cultured together. In some embodiments, the beads and cells are first concentrated by applying a force, such as a magnetic force, which results in increased ligation of cell surface markers, thereby inducing cell stimulation.

As an example, cell surface proteins can be ligated by allowing paramagnetic beads to which anti-CD3 antibodies and anti-CD28 antibodies have been attached (3 x 28 beads) to come into contact with T cells. In some embodiments, cells (e.g., 10 ⁴ to 10 ⁹ T cells) and beads (e.g., DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a 1:1 ratio) are combined in a buffer, such as PBS (without divalent cations such as calcium and magnesium). Those of ordinary skill in the art will also appreciate what concentration of cells can be used. For example, a target cell may be very rare in a sample, comprising only 0.01% of the sample, or the entire sample (i.e., 100%) may consist of the target cell of interest. Accordingly, any number of cells are within the scope of the present invention. In some embodiments, it may be necessary to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) in order to ensure maximum contact of the cells and particles. For example, in some embodiments, a concentration of about 10 billion cells/mL, 9 billion/mL, 8 billion/mL, 7 billion/mL, 6 billion/mL, 5 billion/mL, or 2 billion cells/mL is used. In some embodiments, more than 100 million cells/mL are used. In some embodiments, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In some embodiments, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In some embodiments, concentrations of 125 or 150 million cells/mL can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may have weak expression of target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and may require their production in some embodiments. For example, the use of a high cell concentration allows for more efficient selection of CD8+ T cells, which are typically characterized by weaker expression of CD28.

In some embodiments, cells transduced with a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, are expanded, e.g., by a method described herein. In some embodiments, the cells are expanded in culture for a period of time that is from several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the cells are expanded for a period of 4 to 9 days. In some embodiments, the cells are expanded for a period of 8 days or less, such as 7, 6, or 5 days. In some embodiments, the cells are expanded in culture for 5 days, and the resulting cells are more active than the same cells expanded in culture for 9 days under the same culture conditions. Activity can be determined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, CAR surface expression, CAR level determined by quantitative PCR, or combinations thereof. In some embodiments, cells, such as the CD19 CAR cells described herein, expanded for 5 days exhibit at least a one-, two-, three-, or four-fold increase in cell doubling rate upon antigen stimulation compared to the same cells expanded in culture for 9 days under the same culture conditions. In some embodiments, cells, such as cells expressing a CD19 CAR described herein, are expanded in culture for 5 days and the resulting cells exhibit higher levels of proinflammatory cytokine production, such as IFNγ and/or GM-CSF levels, compared to the same cells expanded in culture for 9 days under the same culture conditions. In some embodiments, cells, such as cells with a CD19 CAR described herein, expanded for 5 days exhibit at least a one-, two-, three-, four-, five-, ten-fold, or greater increase in pg/mL levels of proinflammatory cytokine production, such as IFNγ and/or GM-CSF levels, compared to the same cells expanded in culture for 9 days under the same culture conditions.

Several stimulation cycles may also be required, resulting in a T cell culture time of 60 days or more. Suitable conditions for culturing T cells include suitable media (e.g., minimal essential medium, α-MEM, RPMI 1640 medium, AIM-V, DMEM, F-12, or X-vivo 15 (Lonza), X-Vivo 20, OpTmizer, and IMDM) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFNγ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other cell growth supplements known to one of skill in the art. Other cell growth supplements include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include, but are not limited to, RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, OpTmizer, and IMDM supplemented with amino acids, sodium pyruvate, and vitamins, serum-free, or supplemented with an appropriate amount of serum (or blood plasma), or a defined set of hormones and/or an amount of cytokine(s) sufficient to allow growth and expansion of T cells. Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in cell cultures that are intended for infusion into a subject. The target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g. 37°C) and atmosphere (e.g. air with 5% CO ₂ ).

In some embodiments, the cells are expanded in a suitable medium (e.g., a medium described herein) containing one or more interleukins that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in cell number over a 14-day expansion period, e.g., as measured by a method described herein, such as flow cytometry. In some embodiments, the cells are expanded in the presence of IL-15 and/or IL-7 (e.g., IL-15 and IL-7).

In some embodiments, the methods described herein, e.g., methods of making cells expressing a CAR, comprise removing regulatory T cells, e.g., CD25+ T cells or CD25 ^high T cells, from a population of cells, e.g., using an anti-CD25 antibody or fragment thereof or a CD25-binding ligand of IL-2. Methods for removing regulatory T cells, e.g., CD25+ T cells or CD25 ^high T cells, from a population of cells are described herein. In some embodiments, the methods, e.g., methods of making, further comprise contacting the population of cells (e.g., a population of cells that has been depleted of regulatory T cells, such as CD25+ T cells or CD25 ^high T cells; or a population of cells that has previously been contacted with an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15 and/or IL-7. For example, a population of cells (e.g., a population that has previously been contacted with an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments, a cell expressing a CAR as described herein is contacted with a composition comprising an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha subunit (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide, e.g., hetIL-15, during the manufacture of a cell expressing the CAR, e.g., ex vivo. In some embodiments, a cell expressing a CAR as described herein is contacted with a composition comprising an IL-15 polypeptide during the manufacture of a cell expressing the CAR, e.g., ex vivo. In some embodiments, a cell expressing a CAR as described herein is contacted with a composition comprising a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide during the manufacture of a cell expressing the CAR, e.g., ex vivo. In some embodiments, a CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during the manufacture of the CAR-expressing cell, such as ex vivo.

In some embodiments, a cell expressing a CAR as described herein is contacted with a composition comprising hetIL-15 during ex vivo expansion. In some embodiments, a cell expressing a CAR as described herein is contacted with a composition comprising an IL-15 polypeptide during ex vivo expansion. In some embodiments, a cell expressing a CAR as described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo expansion. In some embodiments, the contacting results in the survival and proliferation of a subset of lymphocytes, such as CD8+ T cells.

T cells that have been stimulated for different periods of time may exhibit different characteristics. For example, in typical blood products or peripheral blood mononuclear cell apheresis products, the population of helper T cells (TH, CD4+) outnumbers the population of cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors results in a T cell population that is composed primarily of TH cells until approximately days 8-9, whereas after approximately days 8-9 the T cell population contains an increasingly large population of TC cells. Accordingly, depending on the purpose of treatment, it may be advantageous to infuse a subject with a T cell population containing primarily TH cells. Similarly, if an antigen-specific subpopulation of TC cells has been isolated, it may be beneficial to expand this subpopulation to a greater extent.

In addition, in addition to the CD4 and CD8 markers, other phenotypic markers vary significantly but largely reproducibly during the cell expansion process. This reproducibility therefore provides the opportunity to tailor an activated T cell product to specific purposes.

Once the CAR described herein has been constructed, various assays can be used to evaluate the activity of the molecule, such as, but not limited to, the ability of T cells to expand after antigen stimulation, sustained expansion of T cells in the absence of re-stimulation, and forms of anti-cancer activity, in appropriate in vitro and animal models. Assays for evaluating the effects of the CAR of the present invention are described in more detail below.

Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers, for example, as described in paragraph 695 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

CAR Reproduction⁺T cellsin vitroafter stimulation with antigen can be measured using flow cytometry. For example, the CD4 mixture⁺and CD8⁺T cells were stimulated with aAPC with αCD3/αCD28 and then transduced with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include those of the CMV IE, EF-1α, ubiquitin C, or phosphoglycerate kinase (PGK) genes. GFP fluorescence was assessed by flow cytometry at day 6 of culture in CD4 subsets.⁺and/or CD8⁺T cells. See, for example, Miloneet al., Molecular Therapy 17(8): 1453–1464 (2009). As an alternative, a mixture of CD4⁺and CD8⁺T cells were stimulated with αCD3/αCD28-coated magnetic beads on day 0 and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR together with eGFP using the 2A ribosome skipping sequence. Cultures were restimulated with K562 cells,⁺with respect to the cancer-associated antigen described herein (K562 expressing the cancer-associated antigen described herein), wild-type K562 cells (wild-type K562), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 antibody and anti-CD28 antibody (K562-BBL-3/28). Exogenous IL-2 was added to the cultures every other day at 100 IU/ml. GFP⁺T cells are enumerated by flow cytometry using bead counting. See, for example, Miloneet al., Molecular Therapy 17(8): 1453-1464 (2009).

It is also possible to measure the level of resistance of CAR reproduction⁺T cells in the absence of re-stimulation. See, for example, Miloneet al., Molecular Therapy 17(8): 1453–1464 (2009). Briefly, mean T cell volume (fL) was measured on day 8 of culture using a Coulter Multisizer III or later particle counter, Nexcelom Cellometer Vision, or Millipore Scepter, or other cell counter devices following stimulation with αCD3/αCD28-coated magnetic beads on day 0 and transduction with the indicated CAR on day 1.

Animal models can also be used to measure the activity of CAR expressing cells, for example, as described in paragraph 698 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

The dose-dependent response to treatment with a CAR can be assessed, for example, as described in paragraph 699 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

The assessment of cell proliferation and cytokine production has been previously described as described in paragraph 700 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

Cytotoxicity can be assessed using a standard 51Cr release assay, such as described in paragraph 701 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety. Alternative methods without the use of radioactive isotopes can also be used.

Cytotoxicity can also be assessed by measuring changes in the electrical impedance of adherent cells, such as with an xCELLigence real-time cell analyzer (RTCA). In some embodiments, cytotoxicity is measured at multiple time points.

To assess specific migration and proliferation of CAR in tumor-bearing animal models, imaging technologies can be used, such as those described in paragraph 702 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

Other assays, including those described in the Examples section of this document and assays known in the art, may also be used to evaluate the CARs described herein.

Alternatively or in combination with the methods disclosed herein, methods and compositions are disclosed for one or more of detecting and/or quantifying cells expressing a CAR (e.g., in vitro or in vivo (e.g., clinical monitoring)); expanding and/or activating immune cells; and/or CAR-specific selection that involve the use of a CAR ligand. In some embodiments, the CAR ligand is an antibody that binds to a CAR molecule, e.g., binds to an extracellular antigen-binding domain of a CAR (e.g., an antibody that binds to an antigen-binding domain, e.g., an anti-idiotype antibody; or an antibody that binds to a constant region of an extracellular binding domain). In other embodiments, the CAR ligand is a CAR antigen molecule (e.g., a CAR antigen molecule described herein).

In some embodiments, a method for detecting and/or quantifying cells expressing a CAR is disclosed. For example, a CAR ligand can be used to detect and/or quantify cells expressing a CAR in vitro or in vivo (e.g., for clinical monitoring of CAR expressing cells in a patient or dosing a patient). The method includes:

obtaining a CAR ligand (optionally a labeled CAR ligand, such as a CAR ligand that contains a marker, bead, radioactive or fluorescent label);

obtaining a cell expressing a CAR (e.g., obtaining a sample containing cells expressing a CAR, such as a manufactured sample or a clinical sample);

contacting a CAR-expressing cell with a CAR ligand under conditions that allow binding to occur, thereby detecting the level (e.g., quantity) of CAR-expressing cells present. Binding of a CAR-expressing cell to a CAR ligand can be detected using standard techniques such as FACS, ELISA, etc.

In some embodiments, a method for expanding and/or activating cells (e.g., immune effector cells) is disclosed. The method comprises:

obtaining a cell expressing a CAR (e.g., a first cell expressing a CAR or a cell transiently expressing a CAR);

contacting said CAR-expressing cell with a CAR ligand (e.g., a CAR ligand described herein) under conditions that cause immune cell expansion and/or proliferation, thereby providing a population of activated and/or expanded cells.

In certain embodiments, the CAR ligand is present on a substrate (e.g., immobilized on or attached to a substrate, e.g., a non-naturally occurring substrate). In some embodiments, the substrate is a non-cellular substrate. The non-cellular substrate can be a solid support selected from, for example, a plate (e.g., a microtiter plate), a membrane (e.g., a nitrocellulose membrane), an array, a chip, or a bead. In some embodiments, the CAR ligand is present on the substrate (e.g., on the surface of the substrate). The CAR ligand can be immobilized on, attached to, or covalently or non-covalently associated (e.g., cross-linked) to the substrate. In some embodiments, the CAR ligand is attached (e.g., covalently attached) to a bead. In the above embodiments, the immune cell population can be expanded in vitro or ex vivo. The method may further comprise culturing the immune cell population in the presence of the CAR molecule ligand, such as by any of the methods described herein.

In other embodiments, the method of cell proliferation and/or activation further comprises adding a second stimulatory molecule, such as CD28. For example, the CAR ligand and the second stimulatory molecule can be immobilized on a substrate, such as one or more beads, thereby increasing the rate of cell proliferation and/or activation.

In some embodiments, a method of selecting or enriching for a cell expressing a CAR is provided. The method comprises contacting a cell expressing a CAR with a CAR ligand described herein; and selecting the cell based on binding to the CAR ligand.

In yet other embodiments, a method for depleting, reducing the number of, and/or killing cells expressing a CAR is provided. The method comprises contacting a cell expressing a CAR with a CAR ligand as described herein; and targeting the cell based on binding to the CAR ligand, thereby reducing the number of and/or killing the cells expressing the CAR. In some embodiments, the CAR ligand is linked to a toxic agent (e.g., a toxin or a drug that is destructive to cells). In some embodiments, the anti-idiotype antibody may mediate effector cell activity, such as forms of ADCC or ADC activity.

Illustrative CAR antibodies that may be used in the methods disclosed herein are described, for example, in WO 2014/190273 and in Jena et al., "Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T cells in Clinical Trials," PLOS March 2013 8:3 e57838, the contents of which are incorporated by reference.

In some embodiments, the compositions and methods herein are optimized for a particular T cell subset, such as described in U.S. Patent Application Ser. No. PCT/US2015/043219, filed July 31, 2015, the contents of which are incorporated herein by reference in its entirety. In some embodiments, the optimized T cell subsets exhibit increased persistence compared to a control T cell, such as a different T cell type (e.g., CD8+ or CD4+) expressing the same construct.

In some embodiments, the CD4+ T cell comprises a CAR as described herein, wherein the CAR comprises an intracellular signaling domain suitable for a CD4+ T cell (e.g., optimized to, e.g., cause its increased persistence), e.g., an ICOS domain. In some embodiments, the CD8+ T cell comprises a CAR as described herein, wherein the CAR comprises an intracellular signaling domain suitable for a CD8+ T cell (e.g., optimized to, e.g., cause its increased persistence), e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain. In some embodiments, the CAR as described herein comprises an antigen-binding domain as described herein, e.g., is a CAR comprising an antigen-binding domain.

In some embodiments, described herein is a method of treating a subject, such as a subject having cancer. The method comprises administering to said subject an effective amount of:

1) CD4+ T cells containing CAR (CARCD4+), which contains:

an antigen-binding domain, such as the antigen-binding domain described herein;

transmembrane domain and

an intracellular signaling domain, such as the first costimulatory domain, such as the ICOS domain; and

2) CD8 ⁺ T cells containing CAR (CARCD8+), which contains:

an antigen-binding domain, such as the antigen-binding domain described herein;

transmembrane domain and

an intracellular signaling domain, such as a second costimulatory domain, such as the 4-1BB domain, the CD28 domain, or another costimulatory domain other than the ICOS domain;

where CARCD4+ and CARCD8+ are different from each other.

The method optionally further comprises administering:

3) a second CD8+ T cell containing CAR (second CARCD8+), which contains:

an antigen-binding domain, such as the antigen-binding domain described herein;

transmembrane domain and

an intracellular signaling domain, wherein the second CARCD8+ comprises an intracellular signaling domain, such as a costimulatory signaling domain, that is absent from CARCD8+, and optionally lacks an ICOS signaling domain.

Delivery methods using biopolymers

In some embodiments, one or more CAR-expressing cells disclosed herein can be administered or delivered to a subject via a biopolymer scaffold, such as a biopolymer implant. Biopolymer scaffolds can support or enhance the delivery, expansion, and/or spread of the CAR-expressing cells described herein. The biopolymer scaffold comprises a biocompatible (e.g., that does not substantially induce an inflammatory or immune response) and/or biodegradable polymer, which can be naturally occurring or synthetic. Exemplary biopolymers are described, for example, in paragraphs 1004-1006 of international application WO2015/142675, filed March 13, 2015, which is incorporated herein by reference in its entirety.

Pharmaceutical compositions and means for treatment

In some embodiments, the present invention provides a method of treating a patient, comprising administering cells expressing a CAR obtained as described herein, optionally in combination with one or more other therapies. In some embodiments, the present invention provides a method of treating a patient, comprising administering a reaction mixture comprising cells expressing a CAR described herein, optionally in combination with one or more other therapies. In some embodiments, the present invention provides a method of transporting or receiving a reaction mixture comprising cells expressing a CAR described herein. In some embodiments, the present invention provides a method of treating a patient, comprising receiving a cell expressing a CAR that has been obtained as described herein, and further comprising administering the cell expressing the CAR to the patient, optionally in combination with one or more other therapies. In some embodiments, the present invention provides a method of treating a patient, comprising obtaining a cell expressing a CAR described herein, and further comprising administering the cell expressing the CAR to the patient, optionally in combination with one or more other therapies. Another type of therapy might be, for example, anti-cancer therapy such as chemotherapy.

In some embodiments, the CAR-expressing cells described herein are administered to a subject in combination with a molecule that reduces the population of Treg cells. Methods that reduce the number of (e.g., deplete) Treg cells are known in the art and include, for example, CD25 depletion, administration of cyclophosphamide, modulation of GITR function. Without being limited by theory, it is believed that reducing the number of Treg cells in a subject prior to apheresis or prior to administration of a cell expressing a CAR described herein results in a reduction in the number of unwanted immune cells (e.g., Tregs) in the tumor microenvironment and a reduction in the risk of relapse in the subject.

In some embodiments, a therapy described herein, e.g., a cell expressing a CAR, is administered to a subject in combination with a molecule that targets GITR and/or modulates GITR function, such as a GITR antagonist and/or an anti-GITR antibody that provides for depletion of regulatory T cells (Treg). In some embodiments, cells expressing the CARs described herein are administered to a subject in combination with cyclophosphamide. In some embodiments, GITR binding molecules and/or molecules that modulate GITR function (e.g., a GITR agonist and/or an anti-GITR antibody that provides for depletion of Treg) are administered prior to administration of a cell expressing a CAR. For example, in some embodiments, a GITR agonist can be administered prior to apheresis of the cells. In some embodiments, cyclophosphamide is administered to the subject prior to administration (e.g., infusion or reinfusion) of a cell expressing a CAR or prior to apheresis of the cells. In some embodiments, cyclophosphamide and an anti-GITR antibody are administered to the subject prior to administration (e.g., infusion or reinfusion) of a cell expressing a CAR or prior to apheresis of the cells. In some embodiments, the subject has a cancer (e.g., a solid cancer or a hematological cancer such as ALL or CLL). In some embodiments, the subject has CLL. In some embodiments, the subject has ALL. In some embodiments, the subject has a solid cancer, such as a solid cancer described herein. Exemplary GITR agonists include, for example, GITR-based fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, for example, the GITR-based fusion protein described in U.S. Patent No. 6,111,090, European Patent No. 090505B1, U.S. Patent No. 8,586,023, PCT Publication Nos. WO 2010/003118 and 2011/090754, or the anti-GITR antibody described in, for example, U.S. Patent No. 7,025,962, European Patent No. 1,947,183B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent No. EP 1,866,339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO2005/055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCT Publication No. WO99/20758, PCT Publication No. WO2006/083289, PCT Publication No. WO 2005/115451, US Patent No. 7618632 and PCT Publication No. WO 2011/051726.

In some embodiments, a cell expressing a CAR described herein is administered to a subject in combination with a GITR agonist, such as a GITR agonist described herein. In some embodiments, the GITR agonist is administered prior to administration of the cell expressing the CAR. For example, in some embodiments, the GITR agonist can be administered prior to apheresis of the cells. In some embodiments, the subject has CLL.

The methods described herein may further comprise formulating a cell expressing a CAR as a pharmaceutical composition. The pharmaceutical compositions may comprise a cell expressing a CAR, such as a plurality of CAR expressing cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) and preservatives. The compositions may be formulated, for example, for intravenous administration.

In some embodiments, the pharmaceutical composition is substantially free of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual amounts of anti-CD3 antibody/anti-CD28 antibody coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, cellular or plasmid components for vector packaging, bacteria, and fungi, e.g., there are no detectable levels thereof. In some embodiments, the bacterium is at least a bacterium selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes group A.

In the event that an "immunologically effective amount,""anti-cancer effective amount,""cancer inhibiting amount," or "therapeutic amount" is indicated, the exact amount of the compositions to be administered can be determined by a physician taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). In general, it can be noted that a pharmaceutical composition containing immune effector cells (e.g., T cells, NK cells) described herein can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, in some cases from 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values within these ranges. T-cell-based compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques that are well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

Illustrative BCMA/CD19-CAR-T-based pharmaceutical compositions

In some embodiments, the BCMA/CD19 dual-specific CAR T cell composition is produced by co-transduction with two unique vectors. Accordingly, in some embodiments, the cell composition comprises a heterogeneous population of cells. In some embodiments, the heterogeneous cell composition comprises untransduced T cells, mono-BCMA-specific CAR T cells, mono-CD19-specific CAR T cells, and dual-specific CAR T cells expressing both BCMA-specific CAR molecules and CD19-specific CAR molecules. These different cell populations may exhibit different forms of activity in the context of treating a disease in a subject.

Without being bound by theory, it is believed that in the context of multiple myeloma treatment, for example, activation of BCMA-specific CAR-T cells may be a major driver of anti-tumor response in patients, while CD19-specific activity may play a role in eliminating less common CD19-positive tumor cells.

In some embodiments, the cell-based composition can be evaluated to determine the relative percentages of four different cell populations, such as to select a cell-based composition that contains a higher percentage of BCMA-specific CAR cells (e.g., mono-BCMA-specific CAR cells and dual BCMA/CD19 CAR cells) than mono-CD19-specific CAR cells.

In some embodiments, the cell-based composition comprises:

(a) the first population of cells containing CAR for BCMA but not CAR for CD19;

(b) a second population of cells containing a CAR for CD19 but not a CAR for BCMA; and

(c) a third population of cells containing both CAR for BCMA and CAR for CD19.

In some embodiments:

(i) the total number of viable cells in the combined second and third populations is less than or equal to approximately 110% (e.g., less than or equal to approximately 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations;

(ii) the total number of viable cells in the combined first and third populations is greater than or equal to about 90% (e.g., greater than or equal to about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells in the combined second and third populations; and/or

(iii) the total number of viable cells in the combined first and third populations is greater than or equal to approximately 5% (e.g., greater than or equal to approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the total number of viable cells in the population.

In some embodiments, the cell-based composition further comprises a fourth population of cells that do not comprise a CAR. In some embodiments, the cell-based composition comprises a population of cells with a mono-CD19-specific CAR that is less than or equal to 110% (e.g., less than or equal to about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less) of the number of cells with a BCMA-specific CAR (e.g., the total number of cells with a mono-BCMA-specific CAR and cells with a dual BCMA/CD19 CAR). In some embodiments, the cell-based composition comprises a population of mono CD19 CAR+ cells that is between about 45% to about 50% (such as about 47%) of the BCMA-specific CAR T cells. In some embodiments, the cell-based composition comprises a population of mono CD19 CAR+ cells that is between about 60% to about 65% (such as about 63%) of the BCMA-specific CAR T cells. In some embodiments, the cell-based composition comprises a population of mono CD19 CAR+ cells that is between about 50% to about 55% (such as about 53%) of the BCMA-specific CAR T cells. In some embodiments, the cell-based composition comprises a population of mono CD19 CAR+ cells that is about 82% of the BCMA-specific CAR T cells.

In some embodiments, the cell-based composition can be assessed to determine the percentage of CAR-positive viable cells to ensure sufficient dosing. Accordingly, in some embodiments, the cell-based composition comprises a population of BCMA-specific CAR cells (e.g., the total number of mono-BCMA-specific CAR cells and dual BCMA/CD19 CAR cells) that is greater than or equal to about 5% (e.g., greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) of the total number of viable cells in the cell-based composition.

In some embodiments, the BCMA-specific CAR cells are BCMA-specific CAR T cells. In some embodiments, the mono-BCMA-specific CAR cells are mono-BCMA-specific CAR T cells. In some embodiments, the CD19-specific CAR cells are CD19-specific CAR T cells. In some embodiments, the mono-CD19-specific CAR cells are mono-CD19-specific CAR T cells. In some embodiments, the BCMA/CD19 dual CAR cells are BCMA/CD19 dual CAR T cells.

Administration of doses

In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is about 1×10 ⁶ , 1.1×10 ⁶ , 2×10 ⁶ , 3.6×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 1.8×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ or 5×10 ⁸ cells/kg. In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is at least about 1×10 ⁶ , 1.1×10 ⁶ , 2×10 ⁶ , 3.6×10 6 , 5×10 ⁶ , 1×10 ⁷ , 1.8×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2× ^{10 8 or} 5×10 ⁸ cells/kg. In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is up to about 1×10 ⁶ , 1.1×10 ⁶ , 2×10 ⁶ , 3.6×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 1.8×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ or 5×10 ⁸ cells/kg. In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is about 1.1×10 ⁶ -1.8×10 ⁷ cells/kg. In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is about 1×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , or 5×10 ⁹ cells. In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is at least about 1×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , or 5×10 ⁹ cells. In some embodiments, the dosage of CAR cells (e.g., CD19 CAR cells) is up to about 1×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , or 5×10 ⁹ cells.

In some embodiments, the subject may require the administration of activated immune effector cells (e.g., T cells, NK cells), and then sequentially repeat blood collection (or apheresis) is performed, the immune effector cells (e.g., T cells, NK cells) are activated from there, and the activated and expanded immune effector cells (e.g., T cells, NK cells) are reinfused into the patient. This process can be performed multiple times every few weeks. In some embodiments, the immune effector cells (e.g., T cells, NK cells) can be activated from collected blood samples of between 10 cc and 400 cc. In some embodiments, the immune effector cells (e.g., T cells, NK cells) are activated from collected blood samples of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, or 80 cc. cm, 70 cu. cm, 80 cu. cm, 90 cu. cm or 100 cc. cm.

The compositions of the invention may be administered by any convenient route. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally, such as by intradermal or subcutaneous injection. Immune effector cell (e.g., T cell, NK cell) compositions may be injected directly into a tumor, lymph node, or site of infection.

T cell population depletion

In some embodiments, the methods disclosed herein further comprise administering a T cell depleting agent following treatment with a cell (e.g., an immune effector cell as described herein) that results in a reduction in (e.g., depletion of) CAR expressing cells (e.g., cells expressing a CAR for CD19). Such T cell depleting agents can be used to effectively deplete a CAR expressing cell population (e.g., cells expressing a CAR for CD19) to reduce toxicity. In some embodiments, the CAR expressing cells have been made according to a method as described herein, such as having been assayed (e.g., before or after transfection or transduction) according to a method as described herein.

In some embodiments, the T cell depleting agent is administered one, two, three, four, or five weeks after administration of a cell, such as an immune effector cell population, as described herein.

In some embodiments, the agent that depletes a population of T cells is an agent that depletes a population of T cells that express a CAR, such as by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-induced cell death. For example, the cells expressing the CARs described herein can also express an antigen (e.g., a target antigen) that is recognized by molecules that are capable of inducing cell death, such as ADCC or complement-induced cell death. For example, the cells expressing the CARs described herein can also express a target protein (e.g., a receptor) that can be targeted by an antibody or antibody fragment. Examples of such target proteins include, but are not limited to, EpCAM, VEGFR, integrins (e.g., ανβ3, α4, αΙ3/4β3, α4β7, α5β1, ανβ3, αν integrins), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/receptor IgE, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7 and EGFR and truncated variants thereof (e.g., variants that retain one or more extracellular epitopes but lack one or more regions in the cytoplasmic domain).

In some embodiments, a cell expressing a CAR co-expresses a CAR and a target protein, such as naturally expressing the target protein or engineered to express the target protein. For example, a cell, such as a population of immune effector cells, can comprise a nucleic acid (e.g., a vector) comprising a CAR nucleic acid (e.g., a CAR nucleic acid described herein) and a nucleic acid encoding a target protein.

In some embodiments, the T cell depleting agent is a CD52 inhibitor, such as an anti-CD52 antibody molecule, such as alemtuzumab.

In other embodiments, a cell, such as a population of immune effector cells, expresses a CAR molecule described herein (e.g., a CD19 CAR) and a target protein recognized by a T cell depleting agent. In some embodiments, the target protein is CD20. In some embodiments in which the target protein is CD20, the T cell depleting agent is an anti-CD20 antibody, such as rituximab.

In additional embodiments of any of the above methods, the methods further comprise transplanting a cell, such as a hematopoietic stem cell, or bone marrow into the mammal.

In some embodiments, the present invention provides a method for preparing a mammal for cell transplantation. The method comprises administering to the mammal an effective amount of a cell comprising a CAR nucleic acid or polypeptide, such as a CD19 CAR nucleic acid or polypeptide. In some embodiments, the cell transplantation is a stem cell transplantation, such as a hematopoietic stem cell transplantation or a bone marrow transplantation. In other embodiments, preparing a subject for cell transplantation comprises reducing the number of cells expressing a target in the subject, such as normal cells expressing CD19 or cancer cells expressing CD19.

Dosage regimen

In some embodiments, a dose of viable cells expressing a CAR (e.g., viable cells expressing a CAR for CD19, BCMA, CD20, or CD22) or a pharmaceutical composition comprising said cells comprises from about 1×10 ⁶ to about 1×10 ⁸ (e.g., from about 2×10 ⁶ to about 5×10 ⁷ , from about 5×10 ⁶ to about 2×10 ⁷ , from about 1×10 ⁶ to about 1×10 ⁷ , from about 1×10 ⁷ to about 1×10 ⁸ , from about 1×10 ⁶ to about 3×10 ⁶ , from about 2×10 ⁶ to about 4×10 ⁶ , from about 3×10 ⁶ to about 5×10 ⁶ , from about 4×10 ⁶ to about 6×10 ⁶ , from about 5×10 ⁶ to about 7×10 ⁶ , from about 6×10 ⁶ to about 8×10 ⁶ , from about 7×10 ⁶ to about 9×10 ⁶ , from about 8×10 ⁶ to about 1×10 ⁷ , from about 9×10 ⁶ to about 2×10 ⁷ , from about 1×10 ⁷ to about 3×10 ⁷ , from about 2×10 ⁷ to about 4×10 ⁷ , from about 3×10 ⁷ to about 5×10 ⁷ , from about 4×10 ⁷ to about 6×10 ⁷ , from about 5×10 ⁷ to about 7×10 ⁷ , from about 6×10 ⁷ to about 8×10 ⁷ , from about 7×10 ⁷ to about 9×10 ⁷ , from about 8×10 ⁷ to about 1×10 ⁸ , about 1×10 ⁶ , about 2×10 ⁶ , about 3×10 ⁶ , about 4×10 ⁶ , about 5×10 ⁶ , about 6×10 ⁶ , about 7×10 ⁶ , about 8×10 ⁶ , about 9×10 ⁶ , about 1×10 ⁷ , about 2×10 ⁷ , about 3×10 ⁷ , about 4×10 ⁷ , about 5×10 ⁷ , about 6×10 ⁷ , about 7×10 ⁷ , about 8×10 ⁷ , about 9×10 ⁷ or about 1×10 ⁸ ) CAR-positive viable cells (e.g., BCMA-CAR+ T cells). In some embodiments, the dose of viable cells expressing a CAR (e.g., viable cells expressing a CAR for CD19, BCMA, CD20, or CD22) is from about 0.5×10 ⁶ viable cells expressing a CAR to about 1.25×10 ⁹ viable cells expressing a CAR (e.g., from 0.5×10 ⁶ viable cells expressing a CAR to 1.25×10 ⁹ viable cells expressing a CAR). In some embodiments, the dose of viable cells expressing a CAR (e.g., viable cells expressing a CAR for CD19, BCMA, CD20, or CD22) is about 1×10 ⁶ , about 2.5×10 ⁶ , about 5×10 ⁶ , about 1.25×10 ⁷ , about 2.5×10 ⁷ , about 5×10 ⁷ , about 5.75×10 ⁷ , or about 8×10 ⁷ viable cells expressing a CAR.

In some embodiments, the dose is calculated based on the number of viable BCMA-CAR+ T cells (e.g., cells positive for a single CAR for BCMA and cells positive for dual CARs for BCMA+/CD19+) measured by flow cytometry at day 4 (96 hours) post-transduction (day 3 (72 hours) post-harvest), as described herein. In some embodiments, the dose of viable CAR-expressing cells (e.g., a BCMA/CD19 dual-specific CAR-T cell product) comprises from about 5× ¹⁰⁶ to about 2× ¹⁰⁷ CAR-positive viable cells (e.g., BCMA-CAR+ T cells).

In some embodiments, a dose of viable cells expressing a CAR (e.g., viable cells expressing a CAR for CD19, BCMA, CD20, or CD22) or a pharmaceutical composition comprising the cells is administered to the subject in one or more (e.g., 2, 3, 4, or more) doses. In some embodiments, the cells or pharmaceutical composition are administered to the subject in two doses. In some embodiments, the one or more doses comprise a first dose and a second dose, wherein the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the first dose is greater than, equal to, or less than the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the second dose.

In some embodiments, the one or more doses comprise a first dose and a second dose, wherein:

(a) the first dose comprises from about 1×10 ⁶ to about 1×10 ⁷ (e.g., from about 2×10 ⁶ to about 8×10 ⁶ , from about 4×10 ⁶ to about 6×10 ⁶ , from about 1×10 ⁶ to about 5×10 ⁶ , from about 5×10 ⁶ to about 1×10 ⁷ , from about 1×10 ⁶ to about 3×10 ⁶ , from about 2×10 ⁶ to about 4×10 ⁶ , from about 3×10 ⁶ to about 5×10 ⁶ , from about 4×10 ⁶ to about 6×10 ⁶ , from about 5×10 ⁶ to about 7×10 ⁶ , from about 6×10 ⁶ to about 8×10 ⁶ , from about 7×10 ⁶ to about 9×10 ⁶ , from about 8×10 ⁶ to about 1×10 ⁷ , about 1×10 ⁶ , about 2×10 ⁶ , about 3×10 ⁶ , about 4×10 ⁶ , about 5×10 ⁶ , about 6×10 ⁶ , about 7×10 ⁶ , about 8×10 ⁶ , about 9×10 ⁶ , or about 1×10 ⁷ ) of viable CAR-positive cells (e.g., BCMA-CAR+ T cells);

(b) the second dose comprises from about 1×10 ⁷ to about 1×10 ⁸ (e.g., from about 2×10 ⁷ to about 8×10 ⁷ , from about 4×10 ⁷ to about 6×10 ⁷ , from about 1×10 ⁷ to about 5×10 ⁷ , from about 5×10 ⁷ to about 1×10 ⁸ , from about 1×10 ⁷ to about 3×10 ⁷ , from about 2×10 ⁷ to about 4×10 ⁷ , from about 3×10 ⁷ to about 5×10 ⁷ , from about 4×10 ⁷ to about 6×10 ⁷ , from about 5×10 ⁷ to about 7×10 ⁷ , from about 6×10 ⁷ to about 8×10 ⁷ , from about 7×10 ⁷ to about 9×10 ⁷ , from about 8×10 ⁷ to about 1×10 ⁸ , about 1×10 ⁷ , about 2×10 ⁷ , about 3×10 ⁷ , about 4×10 ⁷ , about 5×10 ⁷ , about 6×10 ⁷ , about 7×10 ⁷ , about 8×10 ⁷ , about 9×10 ⁷ or about 1×10 ⁸ ) CAR-positive viable cells (e.g., BCMA-CAR+ T cells);

(c) the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the first dose is no more than 1/X, where X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, of the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the second dose; and/or

(d) the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the first dose is from about 1% to 100% (e.g., from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 10% to about 50%, from about 50% to about 90%, from about 10% to about 30%, from about 20% to about 40%, from about 30% to about 50%, from about 50% to about 70%, from about 60% to about 80%, or from about 70% to about 90%) of the number of CAR-positive viable cells (e.g., BCMA-CAR+ T cells) in the second dose.

In some embodiments, the first dose comprises about 5×10 ⁶ viable CAR-positive cells (e.g., BCMA-CAR+ T cells). In some embodiments, the second dose comprises about 1×10 ⁷ or about 2×10 ⁷ viable CAR-positive cells (e.g., BCMA-CAR+ T cells).

In some embodiments, the dose of CAR-positive cells may be increased relative to the initial dose upon subsequent administration. For example, the patient may receive an initial dose of about 1 x 10 ⁶ to about 1 x 10 ⁷ (e.g., about 5 x 10 ⁶ ) viable CAR-positive cells, and may receive a second dose of about 1 x 10 ⁷ to about 1 x 10 ⁸ (e.g., about 1 x 10 ⁷ or about 2 x 10 ⁷ ) CAR-positive viable cells (e.g., BCMA-CAR+ T cells).

Patient selection

In some embodiments of any of the methods of treating a subject or composition for use disclosed herein, the subject has cancer, such as hematological cancer. In some embodiments, the cancer is selected from lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone cell lymphoma of mucosal lymphoid tissue), marginal zone cell lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone cell lymphoma, splenic lymphoma/leukemia, diffuse small B-cell lymphoma of the splenic red pulp, variant hairy cell leukemia, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, nodular marginal zone cell lymphoma, pediatric nodular marginal zone cell lymphoma, primary cutaneous follicular center cell lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma. In some embodiments, the cancer is relapsed and/or refractory cancer.

In some embodiments of any of the methods of treating a subject or composition for use disclosed herein, the subject has CLL or SLL. In some embodiments, a subject who has CLL or SLL has previously been administered a BTK inhibitor therapy, such as ibrutinib, for at least 1-12 months, such as 6 months. In some embodiments, the BTK inhibitor therapy, such as ibrutinib, is a second-line therapy. In some embodiments, the subject has had a partial response or has had stable disease in response to the BTK inhibitor therapy. In some embodiments, the subject has not responded to the BTK inhibitor therapy. In some embodiments, the subject has developed resistance, such as developed mutations that confer resistance to ibrutinib. In some embodiments, the mutations conferring resistance to ibrutinib include a mutation in the gene encoding BTK and/or the gene encoding PLCg2. In some embodiments, the subject is an adult, such as at least 18 years of age.

In some embodiments of any of the methods of treating a subject or composition for use disclosed herein, the subject has DLBCL, such as relapsed and/or refractory DLBCL. In some embodiments, the subject having DLBCL, such as relapsed and/or refractory DLBCL, has previously been administered at least 2 lines of chemotherapy, such as an anti-CD20 antibody therapy and/or an anthracycline chemotherapy. In some embodiments, the subject has previously received a stem cell therapy, such as an autologous stem cell therapy, and has not responded to the stem cell therapy. In some embodiments, the subject does not meet the criteria for a stem cell therapy, such as an autologous stem cell therapy. In some embodiments, the subject is an adult, such as at least 18 years of age.

Therapeutic use

Diseases and/or disorders associated with MCVA

In one aspect, the present invention provides methods for treating a disease associated with BCMA expression. In one aspect, the present invention provides methods for treating a disease wherein a portion of a tumor is BCMA negative and a portion of the tumor is BCMA positive. For example, a CAR of the present invention is useful for treating subjects who have undergone treatment for a disease associated with increased BCMA expression, wherein the subject who has undergone treatment for elevated BCMA levels exhibits a disease associated with elevated BCMA levels. In some embodiments, a CAR of the present invention is useful for treating subjects who have undergone treatment for a disease associated with BCMA expression, wherein the subject who has undergone treatment for a disease associated with BCMA expression exhibits a disease associated with BCMA expression.

In one embodiment, the present invention provides methods for treating a disease in which BCMA is expressed on both normal cells and cancer cells, but is expressed at lower levels on normal cells. In one embodiment, the method further comprises selecting a CAR of the present invention that binds with an affinity that allows the CAR for BCMA to bind to and kill BCMA-expressing cancer cells, but to kill less than 30%, 25%, 20%, 15%, 10%, 5%, or less of the normal cells expressing BCMA, such as determined by an assay described herein. For example, a killing assay such as flow cytometry using Cr51 CTLs can be used. In one embodiment, a CAR for BCMA has an antigen-binding domain that has a binding affinity with a KD for a target antigen of from ^10-4 M to ^10-8 M, such as from ^10-5 M to ^10-7 M, such as ^10-6 M or ^10-7 M. In one embodiment, the antigen-binding domain for BCMA has a binding affinity that is at least five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1000-fold lower than that of a reference antibody, such as an antibody described herein.

In one aspect, the present invention relates to a vector comprising a CAR for BCMA operably linked to a promoter for expression in mammalian immune effector cells, e.g., T cells or NK cells. In one aspect, the present invention provides a recombinant immune effector cell, e.g., a T cell or NK cell, expressing a CAR for BCMA for use in the treatment of BCMA-expressing tumors, wherein the recombinant immune effector cell (e.g., a T cell or NK cell) expressing a CAR for BCMA is referred to as a cell expressing a CAR for BCMA (e.g., a BCMA-CAR-T cell or an NK cell expressing a CAR for BCMA). In one aspect, a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention is capable of contacting a tumor cell with at least one CAR for BCMA of the present invention expressed on its surface, such that the cell expressing the CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) targets the tumor cell and tumor growth is inhibited.

In one aspect, the present invention relates to a method for inhibiting the growth of a tumor cell expressing BCMA, comprising contacting the tumor cell with a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention, such that the cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) is activated in response to an antigen and targets the cancer cell, wherein tumor growth is inhibited.

In one aspect, the present invention relates to a method of treating cancer in a subject. The method comprises administering to the subject a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention, such that the subject is treated for cancer. An example of a cancer treatable with a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention is a cancer associated with BCMA expression.

The present invention includes a type of cell therapy wherein immune effector cells (e.g., T cells or NK cells) are genetically modified to express a chimeric antigen receptor (CAR), and wherein a cell expressing a CAR for BCMA (e.g., a BCMA-CAR-T cell or an NK cell expressing a CAR for BCMA) is administered by infusion to a recipient in need thereof. The infused cell is capable of killing tumor cells in the recipient. Unlike antibody-based therapeutics, CAR-modified cells, such as T cells or NK cells, are capable of replicating in vivo, resulting in long-term persistence that can provide durable tumor control. In various aspects, the cells (e.g., T cells, NK cells) administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after the cell (e.g., T cells or NK cells) is administered to the patient.

The present invention also includes a type of cell therapy in which immune effector cells (e.g., T cells or NK cells) are modified, for example, by in vitro transcribed RNA, so that they transiently express a chimeric antigen receptor (CAR), and the immune effector cell (e.g., T cell or NK cell) is administered by infusion to a recipient in need thereof. The infused cell is capable of killing tumor cells in the recipient. Thus, in various aspects, the immune effector cells (e.g., T cells or NK cells) administered to a patient are present for less than one month, such as three weeks, two weeks, one week, after the immune effector cell (e.g., T cell or NK cell) is administered to the patient.

Without being limited by theory, it is believed that the anti-tumor immune response elicited by CAR-modified immune effector cells (e.g., T cells or NK cells) may be an active or passive immune response, or may alternatively be due to a direct or indirect immune response. In one aspect, CAR-transduced immune effector cells (e.g., T cells or NK cells) exhibit secretion of specific proinflammatory cytokines and potent cytolytic activity in response to BCMA-expressing human tumor cells, counteract inhibition by soluble BCMA, mediate non-specific cytolysis, and mediate regression of established human tumors. For example, antigen-deficient tumor cells within a heterogeneous region of a BCMA-expressing tumor may be susceptible to indirect destruction by BCMA-redirected immune effector cells (e.g., T cells or NK cells) that have previously reacted against adjacent antigen-positive cancer cells.

In one aspect, the fully human CAR-modified immune effector cells (e.g., T cells or NK cells) of the present invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

For ex vivo immunization, prior to administration of the cell to the mammal, at least one of the following events occurs in vitro: i) expansion of the cells, ii) introduction of the CAR-encoding nucleic acid into the cells, or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed in more detail below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutically beneficial effect. The mammalian recipient can be a human, and the CAR-modified cell can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitor cells described in U.S. Patent No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, so the present invention is not limited to any particular method for ex vivo cell expansion. Briefly, ex vivo culturing and expansion of T cells involves (1) collecting mammalian CD34+ hematopoietic stem and progenitor cells from pooled peripheral blood or bone marrow explants and (2) ex vivo expansion of such cells. In addition to the cellular growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used to cultivate and expand the cells.

In addition to the use of a cellular vaccine for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed to an antigen in a patient.

In general, cells activated and expanded as described herein can be used in the treatment and prevention of diseases occurring in immunocompromised individuals. For example, CAR-modified immune effector cells (e.g., T cells or NK cells) of the present invention are used in the treatment of diseases, disorders, and conditions associated with BCMA expression. In some aspects, the cells of the present invention are used in the treatment of patients at risk of developing diseases, disorders, and conditions associated with BCMA expression. Thus, the present invention provides methods for the treatment or prevention of diseases, disorders, and conditions associated with BCMA expression, comprising administering to a subject in need thereof a therapeutically effective amount of CAR-modified immune effector cells (e.g., T cells or NK cells) of the present invention.

In one aspect, the CAR-expressing cells (e.g., CAR-T cells or CAR-expressing NK cells) of the present invention can be used to treat a proliferative disease such as cancer or a malignancy, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia. In one aspect, the cancer is a hematological cancer. Conditions associated with hematological cancers are types of cancer such as leukemia and malignant lymphoproliferative conditions that affect the blood, bone marrow, and lymphatic system. In one aspect, the hematological cancer is leukemia or a hematological disorder. An example of a disease or disorder associated with BCMA is multiple myeloma (also known as MM) (see Claudio et al.,Blood. 2002, 100(6):2175-86; and Novak et al., Blood. 2004, 103(2):689-94). Multiple myeloma, also known as plasma cell myeloma or Kahler disease, is a cancer characterized by the accumulation of abnormal or malignant plasma B cells in the bone marrow. Often, the cancer cells invade adjacent bone, destroying skeletal structures and leading to bone pain and fractures. Most cases of myeloma are also characterized by the production of paraprotein (also known as M proteins or myeloma proteins), which is an abnormal immunoglobulin produced in excess by the clonal proliferation of malignant plasma cells. Serum paraprotein levels greater than 30 g/L are diagnostic of multiple myeloma according to the diagnostic criteria of the International Multiple Myeloma Study Group (IMWG) (see Kyle et al. (2009) Leukemia. 23:3-9). Other symptoms or signs of multiple myeloma include decreased kidney function or kidney failure, bone lesions, anemia, hypercalcemia, and neurologic symptoms.

Criteria for distinguishing between multiple myeloma and other plasma cell proliferative disorders have been established by the International Multiple Myeloma Study Group (see Kyle et al. (2009), Leukemia. 23:3-9). All three of the following criteria must be met:

- clonal plasma cells of bone marrow ≥ 10%;

- the presence of a monoclonal protein in the blood serum and/or urine (except for patients with true non-secretory multiple myeloma);

- evidence of damage to target organs caused by an underlying disorder of plasma cell proliferation, namely:

o hypercalcemia: serum calcium level ≥ 11.5 mg/100 ml,

o renal failure: serum creatinine level > 1.73 mmol/l,

o anemia: normochromic, normocytic with a hemoglobin level that is > 2 g/100 ml below the lower limit of normal, or with a hemoglobin level < 10 g/100 ml,

o bone lesions: osteolytic lesions, severe osteopenia or pathological fractures.

Other plasma cell proliferative disorders that can be treated with the compositions and methods described herein include, but are not limited to, asymptomatic myeloma ("smoldering" multiple myeloma or smoldering myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, forms of plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloidosis in the form of light chain amyloidosis, and POEMS syndrome (also known as Crowe-Fukasa syndrome, Takatsuki disease, and PEP syndrome).

There are two staging systems used to stage multiple myeloma: the International Staging System (ISS) (see Greipp et al. (2005), J. Clin. Oncol. 23 (15):3412–3420) and the Durie–Salmon staging system (DSS) (see Durie et al. (1975), Cancer 36 (3): 842-854).

The third staging system for multiple myeloma is called the Revised International Staging System (R-ISS) (see Рalumbo A, Avet-Loiseau H, Oliva S, et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2015;33:2863-9, incorporated herein by reference in its entirety). R-ISS stage I includes ISS stage I (serum β2-microglobulin < 3.5 mg/L and serum albumin ≥ 3.5 g/dL), no high-risk CA [del(17p) and/or t(4;14) and/or t(14;16)], and LDH within normal limits (less than the upper limit of the normal range). R-ISS stage III includes ISS stage III (serum β2-microglobulin > 5.5 mg/L) and high-risk CA or high LDH. Stage II R-ISS includes all other possible combinations.

Patient responses can be defined according to the IMWG 2016 criteria described in Kumar S, Paiva B, Anderson KC, et al. International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. The Lancet Oncology;17(8):e328–e346, incorporated herein by reference in its entirety.

Standard treatment for multiple myeloma and related disorders includes chemotherapy, stem cell transplantation (autologous or allogeneic), radiation therapy, and other drug therapies. Drugs commonly used to treat myeloma include alkylating agents (eg, bendamustine, cyclophosphamide, and melphalan), proteasome inhibitors (eg, bortezomib), corticosteroids (eg, dexamethasone and prednisone), and immunomodulators (eg, thalidomide and lenalidomide or Revlimid®), or any combination of these. Bisphosphonate drugs are also often given in combination with standard MM treatments to prevent bone loss. Patients over 65 to 70 years of age are unlikely candidates for stem cell transplantation. In some cases, double autologous stem cell transplantations are possible treatment options for patients under 60 years of age with a suboptimal response to a first transplant. The compositions and methods of the present invention can be administered in combination with any of the currently prescribed treatments for multiple myeloma.

Another example of a disease or disorder associated with BCMA is Hodgkin lymphoma and non-Hodgkin lymphoma (see Chiu et al.,Blood. 2007, 109(2):729-39; He et al. J Immunol.2004, 172(5):3268-79).

Hodgkin lymphoma (HL), also known as Hodgkin's disease, is a cancer of the lymphatic system that originates from white blood cells, or lymphocytes. The abnormal cells that form the lymphoma are called Reed-Sternberg cells. In Hodgkin lymphoma, cancer has spread from one group of lymph nodes to another. Hodgkin lymphoma can be divided into four pathological subtypes based on the morphologic characteristics of the Reed-Sternberg cells and the cellular composition around the Reed-Sternberg cells (determined by lymph node biopsy): HL with nodular sclerosis, mixed cell subtype, lymphocyte-rich or lymphocyte-predominant, and lymphocyte-depleted. Occasionally, Hodgkin lymphoma may also present as nodular lymphocyte-predominant Hodgkin lymphoma or may be unspecified. Symptoms and signs of Hodgkin lymphoma include painless swollen lymph nodes in the neck, armpits, or groin, fever, night sweats, weight loss, fatigue, itchy skin, or abdominal pain.

Non-Hodgkin lymphoma (NHL) is a group of diverse blood cancers that includes any type of lymphoma other than Hodgkin lymphoma. Subtypes of NHL are classified primarily by cell morphologic characteristics, chromosomal aberrations, and surface markers. NHL subtypes (or NHL-associated cancers) include B-cell lymphomas such as, but not limited to, Burkitt lymphoma, chronic lymphocytic B-cell leukemia (B-CLL), prolymphocytic B-cell leukemia (B-PLL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) (e.g., intravascular large B-cell lymphoma and primary mediastinal B-cell lymphoma), follicular lymphoma (e.g., follicular center cell lymphoma, follicular small cleaved-nuclear lymphoma), hairy cell leukemia, high-grade B-cell lymphoma (Burkitt-like), lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia), mantle cell lymphoma, forms of B-cell lymphoma marginal zone cell lymphoma (e.g. extranodal marginal zone cell B-cell lymphoma or mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone cell B-cell lymphoma, and splenic marginal zone cell B-cell lymphoma), plasmacytoma/myeloma, progenitor B-lymphoblastic leukemia/lymphoma (PB-LBL/L), primary central nervous system (CNS) lymphoma, primary intraocular lymphoma, small lymphocytic lymphoma (SLL); and T-cell lymphomas such as, but not limited to, anaplastic large cell lymphoma (ALCL), adult T-cell lymphoma/leukemia (e.g., smoldering, chronic, acute, and lymphomatous), lymphangioma, angioimmunoblastic T-cell lymphoma, forms of cutaneous T-cell lymphoma (e.g., mycosis fungoides, Sezary syndrome, etc.), extranodal natural killer/T-cell lymphoma (nasal type), intestinal T-cell lymphoma of enteropathic type, large granular lymphocyte leukemia, T-lymphoblastic lymphoma/progenitor cell leukemia (T-LBL/L), T-cell chronic lymphocytic leukemia/prolymphocytic leukemia (T-CLL/PLL), and unspecified peripheral T-cell lymphoma. Symptoms and signs of Hodgkin lymphoma include painless swollen lymph nodes in the neck, armpits, or groin, fever, night sweats, weight loss, fatigue, itchy skin, abdominal pain, cough, or chest pain.

Staging of Hodgkin lymphoma and non-Hodgkin lymphoma is the same and refers to the extent to which the cancer cells have spread throughout the body. In stage I, lymphoma cells are present in one group of lymph nodes. In stage II, lymphoma cells are present in at least two groups of lymph nodes, but both groups are on the same side of the diaphragm or in the same piece of tissue or organ, and the lymph nodes next to that organ are on the same side of the diaphragm. In stage III, lymphoma cells are present in lymph nodes on both sides of the diaphragm or in the same piece of tissue or organ next to those lymph node groups or in the spleen. In stage IV, lymphoma cells are found in multiple parts of at least one organ or tissue, or lymphoma cells are present in an organ and in lymph nodes on the other side of the diaphragm. In addition to staging with Roman numerals, stages can also be described using the letters A, B, E, and S, where A denotes patients without symptoms, B denotes patients with symptoms, E denotes patients in whom lymphoma is found in tissues outside the lymphatic system, and S denotes patients in whom lymphoma is found in the spleen.

Hodgkin lymphoma is typically treated with radiation therapy, chemotherapy, or hematopoietic stem cell transplant. The most common therapy for non-Hodgkin lymphoma is the R-CHOP regimen, which consists of four different chemotherapy drugs (cyclophosphamide, doxorubicin, vincristine, and prednisone) and rituximab (Rituxan®). Other therapies commonly used to treat NHL include other chemotherapy drugs, radiation therapy, stem cell transplant (autologous or allogeneic bone marrow transplant), or biologic therapies such as immunotherapy. Other examples of biologic therapies include, but are not limited to, rituximab (Rituxan®), tositumomab (Bexxar®), epratuzumab (LymphoCide®), and alemtuzumab (MabCampath®). The compositions and methods of the present invention can be administered in combination with any of the currently prescribed treatments for Hodgkin's lymphoma or non-Hodgkin's lymphoma.

Waldenstrom's macroglobulinemia (WM), also known as lymphoplasmacytic lymphoma (LPL), has also been found to be associated with BCMA expression (see Elsawa et al., Blood . 2006, 107(7):2882-8). Waldenstrom's macroglobulinemia was previously thought to be related to multiple myeloma, but has more recently been classified as a subtype of non-Hodgkin's lymphoma. WM is characterized by uncontrolled proliferation of B-cell lymphocytes, resulting in anemia and production of excess amounts of the paraprotein or immunoglobulin M (IgM), which thickens the blood and leads to hyperviscosity syndrome. Other symptoms or signs of WM include fever, night sweats, fatigue, anemia, weight loss, lymphadenopathy or splenomegaly, blurred vision, dizziness, nosebleeds, bleeding gums, unusual bruising, renal impairment or failure, amyloidosis, or peripheral neuropathy.

The standard treatment for WM is chemotherapy, particularly rituximab (Rituxan®). Other chemotherapy drugs such as chlorambucil (Leukeran®), cyclophosphamide (Neosar®), fludarabine (Fludara®), cladribine (Leustatin®), vincristine, and/or thalidomide may be used in combination. Corticosteroids such as prednisone may also be given in combination with chemotherapy. Plasmapheresis or plasma exchange is usually used throughout the patient's treatment to relieve some of the symptoms by removing the paraprotein from the blood. In some cases, stem cell transplantation is an option for some patients.

Another example of a disease or disorder associated with BCMA is brain cancer. In particular, astrocytoma or glioblastoma has been found to be associated with BCMA expression (see Deshayes et al,Oncogene. 2004, 23(17):3005-12, Pelekanou et al. PLoS One. 2013, 8(12):e83250). Astrocytomas are tumors that arise from astrocytes, which are a type of glial cell in the brain. Glioblastoma (also known as glioblastoma multiforme or GBM) is the most malignant form of astrocytoma and is considered the most advanced stage of brain cancer (stage IV). There are two variants of glioblastoma: giant cell glioblastoma and gliosarcoma. Other forms of astrocytoma include juvenile pilocytic astrocytoma (JPA), fibrillary astrocytoma, pleomorphic xanthoastrocytoma (PXA), dysembryoplastic neuroepithelial tumor (DNET), and anaplastic astrocytoma (AA).

Symptoms or signs associated with glioblastoma or astrocytoma include increased pressure in the brain, headaches, seizures, memory loss, behavioral changes, movement or sensory loss on one side of the body, speech dysfunction, cognitive impairment, vision problems, nausea, vomiting, and weakness in the arms or legs.

Surgical removal of the tumor (or resection) is the standard treatment to remove as much of the glioma as possible with minimal or no damage to surrounding normal brain tissue. Radiation therapy and/or chemotherapy are often used after surgery to suppress and slow recurrent disease from any remaining cancer cells or associated lesions. Radiation therapy includes whole-brain radiation therapy (traditional external beam radiation therapy), targeted 3-D conformal radiation therapy, and targeted radionuclides. Chemotherapeutic agents commonly used to treat glioblastoma include temozolomide, gefitinib or erlotinib, and cisplatin. Angiogenesis inhibitors such as bevacizumab (Avastin®) are also commonly used in combination with chemotherapy and/or radiation.

Maintenance therapy is also often used to alleviate neurological symptoms and improve neurological function and is used in combination with any of the cancer therapies described herein. Key maintenance agents include anticonvulsants and corticosteroids. Thus, the compositions and methods of the present invention can be used in combination with any type of standard or maintenance therapy for the treatment of glioblastoma or astrocytoma.

Diseases and disorders not related to cancer but associated with BCMA expression can also be treated using the compositions and methods disclosed herein. Examples of diseases and disorders not related to cancer associated with BCMA expression include, but are not limited to, viral infections, such as those caused by HIV; fungal infections, such as those caused by C. neoformans ; irritable bowel syndrome; ulcerative colitis; and disorders related to mucosal immunity.

The CAR-modified immune effector cells (e.g., T cells or NK cells) of the present invention can be administered alone or in a pharmaceutical composition in combination with diluents and/or other components, such as IL-2 or other cytokines, or cell populations.

The present invention provides compositions and methods for treating cancer. In one aspect, the cancer is a hematological cancer, including but not limited to a hematological cancer that is leukemia or lymphoma. In one aspect, CAR expressing cells (e.g., CAR T cells or CAR expressing NK cells) of the present invention can be used to treat forms of cancer and malignancy, such as, but not limited to, for example, forms of acute leukemia, including but not limited to, for example, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL); one or more forms of chronic leukemia, including but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematological cancers or conditions, including but not limited to, e.g., B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," which is a diverse group of hematological conditions united by ineffective formation (or dysplasia) of myeloid blood cells, etc. In addition, a disease associated with the expression of BCMA includes, without limitation, for example, atypical and/or non-classical forms of cancer, malignant neoplasms, precancerous conditions or proliferative diseases characterized by the expression of BCMA.

In some embodiments, the composition described herein can be used to treat a disease including, but not limited to, a plasma cell proliferative disorder, such as asymptomatic myeloma ("smoldering" multiple myeloma or smoldering myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, forms of plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloidosis in the form of light chain amyloidosis, and POEMS syndrome (also known as Crowe-Fukasa syndrome, Takatsuki disease, and PEP syndrome).

In some embodiments, a composition described herein can be used to treat a disease including, but not limited to, cancer, such as a cancer described herein, such as prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, or lung cancer.

The present invention also provides methods for inhibiting the proliferation or reducing a population of cells expressing BCMA, the methods comprising contacting a population of cells comprising a cell expressing BCMA with a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention that binds to the cell expressing BCMA. In a particular aspect, the present invention provides methods for inhibiting the proliferation or reducing a population of cancer cells expressing BCMA, the methods comprising contacting a population of cancer cells expressing BCMA with a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention that binds to the cell expressing BCMA. In one aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing BCMA, the methods comprising contacting the population of cancer cells expressing BCMA with a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention that binds to the cell expressing BCMA. In some aspects, a cell expressing a CAR for BCMA (e.g., a BCMA CAR T cell or an NK cell expressing a CAR for BCMA) of the present invention provides a reduction in the level, number, quantity, or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with myeloid leukemia or other cancer associated with cells expressing BCMA, or in a corresponding animal model, compared to a negative control. In one aspect, the subject is a human.

The present invention also provides methods for preventing, treating and/or managing a disease associated with BCMA-expressing cells (e.g., a hematological cancer or an atypical cancer characterized by BCMA expression), wherein the methods comprise administering to a subject in need thereof a BCMA-expressing CAR-expressing cell (e.g., a BCMA-CAR-T cell or an NK cell expressing a CAR-expressing BCMA) of the present invention that binds to a BCMA-expressing cell. In one aspect, the subject is a human. Non-limiting examples of disorders associated with BCMA-expressing cells include viral or fungal infections and disorders associated with mucosal immunity.

The present invention also provides methods for preventing, treating and/or managing a disease associated with BCMA-expressing cells, the methods comprising administering to a subject in need thereof a BCMA-expressing CAR cell (e.g., a BCMA-CAR-T cell or a BCMA-expressing CAR NK cell) of the present invention that binds to a BCMA-expressing cell. In one aspect, the subject is a human.

The present invention provides methods for preventing the recurrence of a cancer associated with BCMA-expressing cells, the methods comprising administering to a subject in need thereof a BCMA-expressing CAR cell (e.g., a BCMA-CAR-T cell or an NK cell expressing a BCMA-carrier) of the present invention that binds to a BCMA-expressing cell. In one aspect, the methods comprise administering to a subject in need thereof an effective amount of a BCMA-expressing CAR cell (e.g., a BCMA-CAR-T cell or an NK cell expressing a BCMA-carrier) described herein that binds to a BCMA-expressing cell, in combination with an effective amount of another therapy.

Types of combination therapy

The CAR-expressing cell described herein can be used in combination with other known agents and therapies. The CAR-expressing cell described herein and at least one additional therapeutic agent can be administered simultaneously in the same or separate compositions or sequentially. If administered sequentially, the CAR-expressing cell described herein can be administered first and the additional agent can be administered second, or the order of administration can be reversed. The CAR therapy and/or other therapeutic agents, procedures, or methods can be administered during periods of active disease expression or during periods of remission or less active disease expression. The CAR therapy can be administered before another therapy, concurrently with the therapy, after the therapy, or during remission of the disorder. When administered in combination, the CAR therapy and the additional agent (e.g., a second or third agent), or all of them, can be administered in an amount or dose that is higher, lower, or the same as the amount or dose of each agent administered alone, such as as a monotherapy. In some embodiments, the amount or dose of the CAR therapy, the additional agent (e.g., the second or third agent), or all of them administered is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each agent administered alone, e.g., as a monotherapy. In other embodiments, the amount or dose of the CAR therapy, the additional agent (e.g., the second or third agent), or all of them that results in the desired effect (e.g., treating cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dose of each agent administered alone, e.g., as a monotherapy, required to achieve the same therapeutic effect. In further aspects, the CAR expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation therapy, immunosuppressive agents. Exemplary agents and therapies that may be used in combination with the CAR expressing cell described herein are disclosed on pages 266-313 of WO2016164731, which is incorporated herein by reference in its entirety.

Biomarkers for assessing CAR efficacy

In some embodiments, disclosed herein is a method for assessing or monitoring the efficacy of a CAR-expressing cell-based therapy (e.g., a CAR-based therapy for CD19 or BCMA) in a subject (e.g., a subject having a cancer, such as a hematological cancer). The method includes collecting data on the efficacy value of the CAR-therapy, wherein the efficacy value is indicative of the efficacy or suitability of the CAR-expressing cell-based therapy.

In some embodiments, the efficacy value of a CAR therapy in a subject having CLL or SLL comprises an indicator of one, two, three, or all of the following parameters:

(i) mutations in the gene encoding BTK in a sample (e.g., a sample obtained by apheresis or a sample of a manufactured product based on cells expressing CAR);

(ii) mutations in the gene encoding PLCg2 in a sample (e.g., a sample obtained by apheresis or a sample of a manufactured CAR-expressing cell product);

(iii) minimal residual disease, such as assessed by the level and/or activity of CD8, CD4, CD3, CD5, CD19, CD20, CD22, CD43, CD79b, CD27, CD45RO, CD45RA, CCR7, CD95, LAG3, PD-1, TIM-3, and/or CD81; or assessed by immunoglobulin deep sequencing; in a sample (e.g., an apheresis sample or a tumor sample from the subject); or

(iv) the level or activity of one, two, three, four, five, six, seven, eight, nine, ten or all of the cytokines selected from IFN-g, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-a, IP-10, MCP1, MIP1a, in a sample, such as a sample obtained by apheresis, from a subject.

In some embodiments, the efficacy measure of a CAR therapy in a subject having DLBCL, such as relapsed and/or refractory DLBCL, comprises an indicator of one or both of the following parameters:

(i) minimal residual disease, such as assessed by the level and/or activity of CD8, CD4, CAR19, CD3, CD27, CD45RO, CD45RA, CCR7, CD95, LAG3, PD-1 and/or TIM-3; or assessed by immunoglobulin deep sequencing; in a sample (e.g., an apheresis sample or a tumor sample from the subject); or

(ii) the level or activity of one, two, three, four, five, six, seven, eight, nine, ten or all of the cytokines selected from IFN-g, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-a, IP-10, MCP1, MIP1a, in a sample (e.g., an apheresis sample from a subject).

In other embodiments, the efficacy value of the CAR therapy agent further comprises an indicator of one, two, three, four, five, six or more (all) of the following parameters:

(i) the level or activity of one, two, three or more (e.g., all) of the resting T _EFF cells, resting T _REG cells, younger T cells (e.g., naive T cells (e.g., naive CD4 or CD8 T cells, naive gamma/delta T cells)), or stem memory T cells (e.g., stem memory CD4 or CD8 T cells or stem memory gamma/delta T cells), or early memory T cells, or a combination thereof in a sample (e.g., a sample obtained by apheresis or a sample of a manufactured CAR-expressing cell product);

(ii) the level or activity of one, two, three or more (e.g., all) of the activated T _EFF cells, activated T _REG cells, aged T cells (e.g., aged CD4 or CD8 cells), or late memory T cells, or a combination thereof, in a sample (e.g., a sample obtained by apheresis or a sample of a manufactured CAR-expressing cell product);

(iii) a level or activity of an immune cell exhaustion marker, such as one, two or more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3, TIGIT, and/or LAG-3), in a sample (e.g., an apheresis sample or a manufactured CAR-expressing cell product sample). In some embodiments, the immune cell has an exhaustion phenotype, such as co-expressing at least two exhaustion markers, such as co-expressing PD-1 and TIM-3. In other embodiments, the immune cell has an exhaustion phenotype, such as co-expressing at least two exhaustion markers, such as co-expressing PD-1 and LAG-3;

(iv) the level or activity of CD27 and/or CD45RO- (e.g., CD27+ CD45RO-) immune effector cells, e.g., in the CD4+ or CD8+ T cell population, in a sample (e.g., an apheresis sample or a manufactured CAR-expressing cell product sample);

(v) the level or activity of one, two, three, four, five, six, seven, eight, nine, ten, eleven or all of the biomarkers selected from CCL20, IL-17a, IL-6, PD-1, PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1;

(vi) the level or activity of cytokines (e.g., the quality of the cytokine repertoire) in a sample of a CAR-expressing cell-based product, such as a sample of a CLL-1-expressing cell-based product; or

(vii) the transduction efficiency of the CAR expressing cell in a sample of the manufactured CAR expressing cell based product.

In some embodiments of any of the methods disclosed herein, the CAR-expressing cell-based therapy comprises a plurality (e.g., a population) of CAR-expressing immune effector cells, such as a plurality (e.g., a population) of T cells or NK cells or a combination thereof. In some embodiments, the CAR-expressing cell-based therapy is a CD19 CAR-based therapy.

In some embodiments of any of the methods disclosed herein, a measure of one or more parameters disclosed herein is obtained from an apheresis sample taken from a subject. The apheresis sample can be assessed prior to infusion or reinfusion.

In some embodiments of any of the methods disclosed herein, a measure of one or more parameters disclosed herein is obtained from a tumor sample taken from a subject.

In some embodiments of any of the methods disclosed herein, a measure of one or more parameters disclosed herein is obtained from a sample of a manufactured CAR-expressing cell product, such as a sample of a CD19 CAR-expressing cell product. The manufactured CAR-expressing cell product can be assessed prior to infusion or reinfusion.

In some embodiments of any of the methods disclosed herein, the subject is assessed prior to, during, or after receiving a CAR-expressing cell-based therapy.

In some embodiments of any of the methods disclosed herein, a profile of one or more of gene expression, flow cytometry, or protein expression is assessed based on one or more of the parameters disclosed herein.

In some embodiments of any of the methods disclosed herein, the method further comprises identifying the subject as a responder, a non-responder, a relapser, or a non-relapser based on a measure of one or more of the parameters disclosed herein.

In some embodiments of any of the methods disclosed herein, a patient who responds to treatment, such as a patient who has a complete response to treatment, has or is identified as having a higher, such as a statistically significantly higher, percentage of CD8+ T cells compared to a reference value, such as the percentage of CD8+ T cells in a patient who does not respond to treatment.

In some embodiments of any of the methods disclosed herein, a responding patient, such as a complete responder, has or is identified as having a higher percentage of CD27+ CD45RO- immune effector cells, such as in the CD8+ population, compared to a reference value, such as the number of CD27+ CD45RO- immune effector cells in a non-responding patient.

In some embodiments of any of the methods disclosed herein, a patient who responds to treatment, such as a patient who has a complete response to treatment or a patient who has a partial response to treatment, has or is identified as having a higher, such as a statistically significantly higher, percentage of CD4+ T cells compared to a reference value, such as the percentage of CD4+ T cells in a patient who does not respond to treatment.

In some embodiments of any of the methods disclosed herein, a responder to treatment, such as a complete responder, has or is identified as having a higher percentage of one, two, three or more (e.g., all) of the resting T _EFF cells, resting T _REG cells, younger T cells, or early memory T cells, or a combination thereof, compared to a reference value, such as the number of resting T _EFF cells, resting T _REG cells, younger T cells, or early memory T cells in a non-responder.

In some embodiments of any of the methods disclosed herein, the non-responding patient has or is identified as having a higher percentage of one, two, three or more (e.g., all) of the activated T _EFF cells, activated T _REG cells, older T cells (e.g., older CD4 or CD8 cells), or late memory T cells, or a combination thereof, compared to a reference value, such as the number of activated T _EFF cells, activated T _REG cells, older T cells (e.g., older CD4 or CD8 cells), or late memory T cells in a responding patient.

In some embodiments of any of the methods disclosed herein, the non-responding patient has or is identified as having a higher percentage of an immune cell exhaustion marker, such as one, two or more immune checkpoint inhibitors (e.g., PD-1, PD-L1, TIM-3, TIGIT and/or LAG-3). In some embodiments, the non-responding patient has or is identified as having a higher percentage of immune effector cells expressing PD-1, PD-L1 or LAG-3 (e.g., CD4+ T cells and/or CD8+ T cells) (e.g., CD4+ cells and/or CD8+ T cells expressing a CAR), compared to the percentage of immune effector cells expressing PD-1 or LAG-3 in the responding patient.

In some embodiments, the non-responding patient has or is identified as having a higher percentage of immune cells having an exhaustion phenotype, such as immune cells that co-express at least two exhaustion markers, such as co-expressing PD-1, PD-L1, and/or TIM-3. In other embodiments, the non-responding patient has or is identified as having a higher percentage of immune cells having an exhaustion phenotype, such as immune cells that co-express at least two exhaustion markers, such as co-expressing PD-1 and LAG-3.

In some embodiments of any of the methods disclosed herein, the non-responding patient has or is identified as having a higher percentage of PD-1/PD-L1+/LAG-3+ cells in the population of CAR expressing cells (e.g., the population of CLL-1-CAR+ cells) compared to a responding patient (e.g., a complete responder) with a CAR expressing cell-based therapy.

In some embodiments of any of the methods disclosed herein, a patient responding to treatment (e.g., a patient with a complete or partial response to treatment) has one, two, three or more (or all) of the following profiles:

(i) there is a higher number of CD27+ immune effector cells compared to a reference value, such as the number of CD27+ immune effector cells in a non-responding patient;

(ii) there is a higher CD8+ T cell count compared to a reference value, such as the CD8+ T cell count in a non-responding patient;

(iii) there is a lower number of immune cells expressing one or more immune checkpoint inhibitors, such as immune checkpoint inhibitors selected from PD-1, PD-L1, LAG-3, TIM-3 or KLRG-1 or a combination thereof, compared to a reference value, such as the number of cells expressing one or more immune checkpoint inhibitors in a non-responding patient; or

(iv) there is a greater number of one, two, three, four or more (all) of resting T _EFF cells, resting T _REG cells, naive CD4 cells, unstimulated memory cells or early memory T cells or a combination thereof compared to a reference value, such as the number of resting T _EFF cells, resting T _REG cells, naive CD4 cells, unstimulated memory cells or early memory T cells in a non-responding patient.

In some embodiments, a subject that is a responder, non-responder, relapsed, or non-relapsed patient identified by the methods set forth herein may be further evaluated according to clinical criteria. For example, a complete responder is or is identified as a subject that has a disease, such as cancer, that exhibits a complete response to treatment, such as a complete remission. A complete response can be identified, for example, using the NCCN Guidelines® or the 2018 International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines as disclosed in Hallek M et al., Blood (2018) 131:2745-2760 "iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL," the entire contents of which are incorporated herein by reference in their entirety. A partial responder is a subject or identified as a subject who has a disease, such as cancer, that is showing a partial response to treatment, such as a partial remission. A partial response may be identified, for example, using the 2018 NCCN Guidelines® or iwCLL criteria, as described herein. A non-responder is a subject or identified as a subject who has a disease, such as cancer, that is not showing a response to treatment, such as the patient has stable disease or progression of disease. A non-responder may be identified, for example, using the 2018 NCCN Guidelines® or iwCLL criteria, as described herein.

Alternatively or in combination with the methods disclosed herein, taking into account the specified value, one, two, three, four or more of the following are performed:

administering, for example, to a responding patient or a non-relapsed patient, a CAR-expressing cell-based therapy;

administration of altered doses of CAR cell-based therapy;

changes in the schedule or timing of administration of the CAR cell-based therapy;

administering, for example, to a non-responding patient or a patient with a partial response to treatment, an additional agent in combination with a CAR-expressing cell-based therapy, for example, an immune checkpoint inhibitor, for example, an immune checkpoint inhibitor described herein;

administering to a non-responding patient or a patient with a partial response to treatment a therapy that provides an increase in the number of younger T cells in the subject, prior to treatment with a therapy based on cells expressing CAR;

modifications to the method of making a CAR-expressing cell-based therapy, such as enriching for younger T cells prior to administration of a CAR-encoding nucleic acid or increasing transduction efficiency, such as in a subject identified as a non-responder or a partial responder;

the introduction of an alternative therapy, for example, to a patient who is not responding to treatment, or to a patient who has had a partial response to treatment, or to a patient who has relapsed; or,

if the subject is a non-responder or relapsed patient or is identified as such, reducing the population T_REG-cells and/or the level of expression of the T gene signature_REG, For example, via one or more of CD25 depletion, cyclophosphamide administration, anti-GITR antibody, or a combination thereof.

EXAMPLES

The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Accordingly, the present invention should in no way be construed as limited by the following examples, but, on the contrary, should be construed as embracing any and all modifications that become apparent as a consequence of the teachings given herein.

Example 1. In vitro characterization of CAR for human BCMA

A panel of fully human single-chain variable fragments (scFv) were cloned into lentiviral CAR expression vectors with CD3 zeta chain and 4-1BB stimulatory molecules: R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03, and Hy52. Constructs were initially screened using an automated reporter cell assay, followed by selection of optimal clones based on expression on primary T cells and the quantity and quality of effector T cell responses (“BCMA-CAR-T” or “BCMA-CAR-T cells”) to BCMA-expressing targets (“BCMA+” or “BCMA-positive”). Responses involving effector T cells include, but are not limited to, expansion, proliferation, duplication, cytokine production, and target cell killing or cytolytic activity (degranulation) in cells.

Production of a lentiviral vector encoding a CAR for BCMA

All the above lentiviral transfer vectors encoding scFv were used to generate genomic material packaged into VSVg pseudotyped lentiviral particles. The lentiviral transfer vector DNA encoding CAR was mixed with the three packaging components VSVg, gag/pol and rev in combination with lipofectamine reagent to transfect Lenti-X 293T cells (Clontech) followed by medium change after 12-18 h. After 30 h of medium change, the medium was collected, filtered and stored at -80°C.

JNL with CAR for BCMA and screening assay in JNL reporter cells using an automated system

For the reporter cell assay, lentivirus encoding the CAR for BCMA was produced in HEK293 cells at two different cell densities (40,000 cells (1x H293) or 80,000 cells (2x H293)) in an automated small-scale mode in 96-well plates, with the virus-containing supernatant collected 48 h post-transfection and used fresh without freezing to transduce the Jurkat reporter T cell line. The Jurkat NFAT-luciferase reporter cell line (JNL) is derived from the Jurkat acute T-cell leukemia cell line. The line was modified to express luciferase under the control of the nuclear factor of activated T (NFAT) response element. For transduction with BCMA CAR, 10,000 JNL cells/well of a 96-well plate were transduced with 50 μl of fresh virus-containing supernatant filtered through a 45 μm filter. Plates were cultured for 5 days before co-culture with target cells.

To evaluate the functional ability of BCMA CAR to activate JNL cells, they were co-cultured with target cancer cells at different effector to target cell ratios (E:T ratios) to detect their activation by quantifying luciferase expression. The scFv-based CARs R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03, and Hy52 were evaluated. JNL cells with CD19 CAR were used as a target-specific control, and medium alone without target cells served as a negative control.

The above 5-day-old CAR-transduced JNL cells were co-cultured with the BCMA-positive multiple myeloma (MM) cell line KMS11 or the NALM6 acute lymphocytic leukemia cell line, which served as a BCMA-negative control. The remaining JNL CAR T cells were assessed for BCMA CAR expression by flow cytometry. Co-cultures were prepared in 384-well plates at effector-to-target (E:T) ratios of 4:1, 2:1, 1:1, and 0.5:1 and incubated for 24 h, after which luciferase expression was quantified on activated JNL CAR T cells using the Bright-Glo™ Luciferase Assay System (Promega, Madison, WI). The amount of light emitted from each well (luminescence) was a direct measure of JNL activation by the respective CAR. JNL cells were considered activated if the luminescence level was equal to or greater than twice that of UTD cells. The BCMA+ cell line KMS11 activated JNL cells expressing R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03, and Hy52 (Fig. 1A and 1C). None of the BCMA CARs showed activation by the BCMA-negative NALM6 line (Fig. 1E and 1F). Medium alone without target cells did not activate any of the CAR-transduced JNL tested (Fig. 1G and 1H). FACS analyses demonstrated that CAR expression for BCMA was detected to varying degrees in transduced JNL; % CAR generally correlated positively with JNL activation by KMS11 cells for the most active CAR-T JNL (Fig. 1B and 1D).

Obtaining BCMA-CAR-T cells

The following 8 CARs were selected for analysis of CAR expression, stability, and potency in primary T cells: R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03, and Hy52. BCMA CAR T cells were generated starting from blood of healthy apheresis donors whose T cells (CD4+ and CD8+ lymphocytes) were obtained by negative selection for CD3+ T cells. These cells were activated by adding anti-CD3/CD28 beads (Dynabeads® Human T-Expander CD3/CD28, Thermo Fisher Scientific) at a ratio of 1:3 (T cells to beads) in T cell medium (RPMI-1640, 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 1x penicillin/streptomycin, 100 μM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 55 μM 2-mercaptoethanol). T cells were cultured at 0.5 x 10 ⁶ T cells in 1 ml medium per well of a 24-well plate at 37°C, 5% CO ₂ . After 24 hours, when T cells had undergone blast formation, T cells were transduced with a virus encoding CAR for BCMA at a multiplicity of infection (MOI) of 5. T cells began dividing with a logarithmic growth pattern monitored by measuring the number of cells per ml, and T cells were diluted with fresh medium every two days, pelleted, and collected for further analysis on day 9. Aliquots of T cells were stained for CAR expression by flow cytometry on days 5 and 9 on a FACS Fortessa (BD). All BCMA CAR T cells were produced under research-grade (i.e., non-clinical grade) manufacturing conditions.

Surface expression of CAR for BCMA and its stability were assessed by measuring %CAR and MFI (mean fluorescence intensity) at day 5 and day 9 using flow cytometric analysis of cells stained with rBCMA_Fc-AF647 (Fig. 2 and Table 27). CAR for BCMA expression in the final product at day 9 varied across constructs, ranging from 18% to 42.4%, and MFI from 672 to 5238. Constructs from the PALLAS-derived clones R1F2, R1B6, and R1G5 and the hybridoma clone Hy03 exhibited a loss of CAR from ~30% to 50% from day 5 to day 9, whereas PI61, B61-10 and -02, and Hy52 were relatively stable in terms of percentage CAR expression, although all CAR constructs exhibited a decrease in MFI from day 5 to day 9, likely due to the smaller size of T cells in their resting stage at day 9. Cell counts in CAR T cell cultures indicated no detectable negative effect of CAR for BCMA carrying human scFv on the ability of cells to proliferate normally compared to with untransduced T cells ("UTD").

Table 27. CAR expression analysis CAR design Titer % CAR on T cells MFI CAR on T cells Day 5 Day 9 Day 5 Day 9 R1B6 2.68E+08 40.0 27.4 24,420 2,367 R1F2 3.60E+08 48.8 22.7 4,716 672 R1G5 2.27E+08 52.0 30.7 29,113 5,238 PI61 1.71E+08 47.4 42.3 24,360 2,099 B61-10 7.06E+07 41.1 30.3 27,298 3,288 B61-02 8.16E+07 33.5 23.6 29,113 3,471 Hy03 4.96E+07 33.7 18.1 9,463 929 Hy52 7.03E+07 35.1 36.1 33,694 2,859

Evaluation of functional properties of CAR-redirected T cells for BCMA

To assess the functionality of BCMA CAR T cells, co-cultures were prepared with BCMA-positive and BCMA-negative cancer cell lines. CAR T cells were thawed, counted, and co-cultured with target cells to record their killing and cytokine secretion capabilities. The BCMA CAR clones tested were R1B6, R1F2, R1G5, B61-02, B61-10, PI61, Hy03, and Hy52. Untransduced T cells (UTD) were used as control non-targeting T cells.

CAR-T cell killing was performed by co-culture of CAR-T cells with KMS11-Luc and NALM6-Luc target cells at various E:T ratios for 20 h. CAR-T cell populations were normalized to equivalent percentages of CAR-positive cells before plating. The cytokine IFNγ was measured in supernatants of 20-h CAR-T cell co-cultures with target cells at an effector:target ratio of 2.5:1 using Meso Scale Discovery (MSD; Gaithersburg, MD), and results for each cytokine were calculated in pg/mL using known standards. All assays were performed in duplicate from a single donor cell source. Killing data showed that all BCMA CAR clones efficiently killed KMS11 cancer cells (Fig. 3A). Control NALM6 target cells were not killed by any of these BCMA-specific CARs (Fig. 3B). The ability of these CARs to produce IFNγ when cultured with KMS11 was also tested (Fig. 3C). BCMA CARs R1F2, R1G5, and PI61 resulted in the highest amounts of IFNγ. The levels of cytokines produced by BCMA CAR-Ts following exposure to control NALM6 cells were low (Fig. 3C), indicating a lack of non-specific activation by BCMA CARs.

Conclusions

Novel BCMA-binding scFvs were tested as part of CAR T cells. Eight CARs were analyzed in JNL reporter assays as well as in primary T cells: R1B6, R1F2, R1G5, B61-02, B61-10, PI61, Hy03, and Hy52. All eight CAR T cell types demonstrated target-specific killing. T cells expressing R1F2, R1G5, or PI61 produced the highest amounts of IFNγ in the presence of target cells. Overall, transfer of BCMA CARs into primary T cells induced CAR reactivity to BCMA but did not induce off-target function.

Example 2. Dual CAR expression and in vitro activity of CAR-T with dual specificity to BCMA/CD19

A series of bicistronic constructs containing two complete CAR (chimeric antigen receptor) chains, one targeting BCMA and the other targeting CD19, were constructed in a lentiviral vector (Table 28). CAR expression is driven by the EF1alpha promoter. These CARs contain a series of human single-chain variable fragments (scFv) targeting BCMA (duBCMA.4, PI61, R1G5, and R1B6). All constructs used the same engineered humanized scFv targeting CD19. At the N-terminus of each scFv, a CD8alpha-derived signal peptide targets the CAR to the secretory pathway. This signal peptide is predicted to be cleaved cotranslationally and therefore will be absent from the mature form of the CAR displayed on the cell surface. At the C-terminus of each scFv is a hinge region and a CD8alpha transmembrane domain fused to a 4-1BB intracellular domain, followed by a CD3zeta intracellular domain. A linker (GSG (SEQ ID NO: 206)) was designed between the two CARs, more specifically between the last amino acid of the first CD3-zeta domain and the signal peptide of the subsequent CAR, followed by the self-cleaving peptide 2A from porcine teschovirus-1 2A (i.e., the P2A sequence). Other linkers and/or self-cleaving peptides can also be used. This design allows the expression of two independent CARs from a single mRNA transcript. The DNA sequences encoding the overlapping regions between the two CARs (signal peptide, hinge region, transmembrane domain, 4-1BB, and CD3-zeta) are different from each other to minimize potential recombination.

Table 28. Brief description of structures Design No. Description 234 duCD19.1-duBCMA.4 235 duBCMA.4-duCD19.1 236 R1G5-duCD19.1 237 R1B6-duCD19.1 238 PI61-duCD19.1 244 Mono-duCD19.1 245 Mono-duBCMA.4

The constructs were used to generate vector material that was used to infect primary human T cells. CAR expression was assessed by flow cytometry. The quantity and quality of effector T cell responses ("BCMA/CD19 dual-specific CAR T cells" or "T cells") to BCMA-expressing ("BCMA+" or "BCMA-positive") and CD19+ target tumors were also measured. Effector T cell responses include, but are not limited to, cell expansion, proliferation, duplication, cytokine production, and target cell killing or cytolytic activity (degranulation).

Obtaining lentivirus and determining titer

The five constructs encoding dual BCMA/CD19 CARs described above were used to generate genomic material packaged into VSVg-pseudotyped lentiviral particles. Two constructs were used as controls: one encoding a mono-CAR directed against BCMA (duBCMA.4) and one encoding a mono-CAR directed against CD19. All seven constructs were constructed in the same plasmid backbone. Each of these DNAs was mixed with the three packaging components VSVg, gag/pol and rev in combination with lipofectamine reagent to transfect Lenti-X 293T cells (Clontech) followed by a medium change 12-18 h later. Thirty hours after the medium change, the medium was collected, filtered and stored at -80°C.

Lentivirus titer was determined by assessing the surface expression of dual CARs for BCMA/CD19 on transduced Sup-T1 cells using Alexa-647-conjugated recombinant human BCMA protein with an Fc tag (BCMA-Fc) and an anti-idiotypic antibody to duCD19.1 (PE). Sup-T1 cells were transduced with virus-containing supernatants in 3-fold serial dilutions from a starting dilution of 1:3. The percentage of CAR-expressing cells (CAR+ cells) was assessed after four days. Viral titer was calculated using the upstream CAR-positive or double CAR-positive population according to the following formula:

(% CAR+) x (number of Sup-T1 cells seeded) x (dilution) / (amount of virus (ml)).

The virus titer was calculated from the central point of the highest dilution in the linear range giving from 5 to 25% CAR+ cells (Table 29).

Table 29. Titers in supT1 Design No. Based on CAR+ over CAR (a) Based on double+ by CAR (b) 234 5.39E+07 1.91E+07 235 4.41E+08 1.15E+08 236 2.14E+08 1.17E+08 237 1.97E+08 8.51E+07 238 1.85E+08 5.66E+07

Production of BCMA/CD19 CAR T cells using the traditional 10-day production method

Dual-specific BCMA/CD19 CAR T cells or mono-BCMA or mono-CD19 CAR T cells were generated using primary human T cells (CD4+ and CD8+ lymphocytes) obtained by negative selection or positive selection with Prodigy in blood from healthy apheresis donors. Prior to transduction, T cells were activated with anti-CD3/CD28 beads (Dynabeads® Human T-Expander CD3/CD28, Thermo Fisher Scientific) at a 1:3 ratio (T cells to beads) in T cell medium (RPMI-1640, 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 1x penicillin/streptomycin, 100 μM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 55 μM 2-mercaptoethanol). Cells were cultured at a density of 0.5 x 10 ⁶ /mL medium per well in a 24-well plate at 37°C, 5% CO ₂ . After 24 hours, when T cells had blasted, they were transduced with viruses at a multiplicity of infection (MOI) of 5 based on the titer of the upstream CAR. T cells began dividing in a logarithmic growth pattern monitored by measuring cells per ml, and T cells were diluted with fresh medium every two days, debeaded, and collected for further analysis at day 8 or later depending on cell size. Aliquots of T cells were stained for CAR expression by flow cytometry on days 7 or 8 on a FACS Fortessa (BD).

Surface expression of BCMA CAR and CD19 CAR was assessed by measuring % CAR and MFI (mean fluorescence intensity) at day 8 using flow cytometry assays of cells stained with rBCMA_Fc-AF647 and duCD19.1 anti-idiotype antibody (PE) (Figs. 4A-4C). Three vectors encoding dual CARs containing R1G5, R1B6, or PI61 resulted in similar percentages of CAR double-positive cells (~10%), with construct 236 (R1G5/duCD19.1) exhibiting the highest MFI for BMCA and CD19 CAR (Fig. 4A). Mono-duCD19.1 (construct 244) exhibited the highest MFI for CAR19 among the different constructs (Fig. 4B).

Evaluation of the functional properties of a BCMA/CD19 dual CAR T cell product in D8 in vitro and in vivo

To evaluate the antitumor efficacy of each CAR, BCMA/CART dual-specific CAR-T cells were cocultured with BCMA-positive (KMS11), CD19-positive (NALM6), and BCMA/CD19-negative cancer cell lines (CD19KO; derived from NALM6). CAR-T cells were thawed, counted, and cocultured with target cells to detect their killing and cytokine secretion capabilities. Untransduced T cells (UTD) were used as control non-targeting T cells.

In vitro cytotoxicity assays were performed by co-culture of CAR-T cells with luciferase-expressing target cells at various E:T ratios for 20 h. CAR-T cell populations were normalized to equivalent percentages of CAR-positive cells prior to seeding according to the CAR targeting the respective tumor (i.e., normalized to CAR for BCMA when CAR-T was co-cultured with KMS11 cells; and normalized to CAR19 when CAR-T was co-cultured with NALM6). The cytokine IFNγ was measured in supernatants of 20-h co-cultures corresponding to a target ratio of 1.25:1 or 2.5:1 (CAR-T:target cells) using Meso Scale Discovery (MSD; Gaithersburg, MD). Results were calculated in pg/mL using known standards. All assays were performed in duplicate from a single donor cell source. Killing data showed that all dual CAR clones were effective against BCMA-positive KMS11 cells and CD19-positive Nalm6 cells (Figs. 5A and 5B, respectively). Only background killing was observed against BCMA/CD19-negative tumor cells (Fig. 5C). The ability of these dual CAR cells to produce IFNγ when co-cultured with KMS11 or Nalm6 was similar when assessed using two different E:T ratios (Figs. 6A-6D).

The antitumor activity of these CAR-Ts at D8 in vivo was analyzed using a mixed xenograft tumor model (5% Nalm6 and 95% KMS11) in NSG mice.

Generation and Measurement of Day 1 BCMA/CD19 Dual-Specific CAR-T Cells Using the Activation-Based Rapid Manufacturing (ARM) Method

In some embodiments, the ARM method was initiated with a frozen or fresh leukapheresis product. After obtaining a sample for counting and QC, the product was connected to a cell sorting device (e.g., a CliniMACS Prodigy device kit) and the program was started. The cells were washed and incubated with microbeads that bind to desired surface markers, such as CD4 and CD8. Bead-labeled cells were selected by passing the cells through a magnetic column. The isolated cells were washed again and the cell media was replaced with separation buffer. The purified T cells were then transferred to culture or cryopreserved for subsequent use. The purity of the isolated T cells was assessed by flow cytometry for a QC step. The cryopreserved cells were thawed, washed in pre-warmed cell media, and resuspended in cell media. Fresh cells were added directly to the culture. Cells were seeded in membrane bioreactors at 0.4-1.2e6 cells/ ^cm2 of membrane, an activating reagent such as beads/polymers containing CD3/CD28 antibodies, nanoparticles or colloidal nanosystems was added and cell media was added in a final volume of 0.25-2 ml/ ^cm2 of membrane. A 24-well GRex was used to study the kinetics of CAR expression in vitro . A GRex 100, flask or CentriCult was used to test the scalability of this fabrication method and to test the antitumor efficacy in vivo . At the time of seeding, cells were transduced with a lentiviral vector encoding a dual CAR for BCMA/CD19 at a multiplicity of infection (MOI) of 1 or 2. The MOI was determined based on the titer of virus produced in SupT1 cells, based on the titer for the CAR engineered above the 2A sequence. After 24 hours, cells were washed to remove unnecessary reagents before staining to measure CAR expression levels by flow cytometry and re-formulated in cryopreservation medium as a “day 1 CAR-T product” for in vivo studies. In all cases, an aliquot of cells was collected 72 hours post-transduction to measure CAR expression kinetics in vitro. Day 1 CAR-T responses include, but are not limited to, cytolytic activity and in vivo expansion.

Figures 7A-7B show the CAR expression pattern for both BCMA and CD19 at 24 and 72 hours post-transduction of primary human T cells made using the ARM method using an MOI of 1 based on the titer in SupT1 determined by expression of the upstream CAR alone. Twenty-four hours post-transduction, flow cytometric analysis showed that the entire live CD3+ T cell population was shifted to the right to varying degrees (Figure 7A). This expression pattern was in contrast to typical flow cytometric histograms of cells transduced to express CAR, where the CAR-positive population is clearly separated from the negative population. These data suggest that "pseudotransduction or transient expression" can be detected using the rBCMA_Fc flow cytometric staining reagent. It has been previously reported that lentiviral pseudotransduction was observed from vector addition for up to 24 hours in CD34+ cells and up to 72 hours in 293 cells (Haas DL, et al. Mol Ther. 2000. 291: 71-80). Integrase-deficient lentiviral vector induced transient eGFP expression for up to 10 days in CD34+ cells and for up to 14 days in 293 cells. Although the observed CAR expression on day 1 after T cell transduction could potentially be explained by pseudotransduction, at day 3, however, two distinct populations emerged, one positive for rBCMA_Fc and the other positive for the anti-idiotypic antibody to duCD19.1 (Fig. 7B and Fig. 7C). Moreover, 20-30% of cells were mono-BCMA CAR positive (CD19 CAR negative) among cells engineered using dual constructs of c235, c236, c237, and c238 (Fig. 7C). Overall, these results demonstrate that both CARs were well expressed in the dual-specificity CAR-T system using R1G5, R1B6, and PI61 in combination with duCD19.1. 72 hours after viral transduction may serve as a surrogate time point to measure CAR expression to guide in vivo dosing strategies.

Evaluation of in vivo functional properties of day 1 BCMA-CAR-T cells treated with ARM

Day 1 CAR-T cells generated by the CentriCult or flask system were studied for their in vivo anti-tumor activity using three mouse models: a KMS-11-luc (BCMA+ CD19-) disseminated multiple myeloma model, a Nalm6-Luc (CD19+ BCMA-) xenograft mouse model, and a mixed model of 95% KMS-luc cells and 5% NALM6-Luc cells. This in vivo study had three objectives: (1) to demonstrate the efficacy of both BCMA and CD19 arms in dual-specificity CAR-T cells; (2) to compare the mixed model (containing BCMA+ tumor cells and CD19+ tumor cells) with a KMS-11-luc (BCMA+ CD19-) multiple myeloma model to understand the potential activation of dual-specificity CAR-T cells through the CD19 target; and (3) test the dual-specificity CAR-T in a Nalm6-only model (CD19+ BCMA-) to examine the activity of the CD19 arm. BLI measurements were performed twice weekly. Peripheral blood was collected on days 6, 13, 20, and 27 for flow cytometry analysis. Plasma was collected on the above days and on day 2 for cytokine analysis. The study design and dosing information are summarized in Table 30.

Table 30. In vivo study design and dosing regimen KMS11 (BCMA) Nalm6 (CD19) A mixture of KMS11 (BCMA) and Nalm6 (CD19), both labeled with Luc c236 1e4, 5e4 1,5e5 1e4, 5e4 c238 1e4, 5e4 1,5e5 1e4, 5e4 Mono-PI61 1e4, 5e4 1e4, 5e4 Mono-CTL119 1,5e5 1e4, 5e4

Dual-specificity CAR-T cells cleared mixed tumors (BCMA+ CD19+) at 5e4 and inhibited, but did not completely eliminate, tumors at 1e4 throughout the study (Fig. 8C). Neither PI61 nor CTL119 could control mixed tumors (Fig. 8C). Both dual c236 and c238 cells demonstrated similar or greater efficacy than their single-specific counterparts in the KMS11 and Nalm6 models (Figs. 8A and 8B). Body weight increased in all three models over the course of the study (Figs. 9A–9C). At the end of the study, a slight decline was observed for KMS11 and NALM6, possibly due to GVHD.

T cell expansion occurred from day 6 to day 20 and then leveled off from day 20 to day 27 (Figs. 10A-10C). Expansion was dose related in all three models (Figs. 11A-11C). The KMS11 model exhibited 3-4 times more expansion than the mixed model at higher doses (Figs. 10A and 10C). Dual c236 and c238 exhibited more expansion than single CAR-T (Figs. 10A-10C).

The overall BCMA-CAR+ percentage peaked at day 13 and then began to decline through day 27 (Figs. 11A-11C). In the mixed model, the 5e4 groups exhibited lower BCMA-CAR+ percentages compared to the 1e4 groups after day 13 (Fig. 11C). The double CAR+ percentage was associated with an influx of the total CD3+ cell population, which could be a possible hallmark of GVHD. Double BCMA/CD19-CAR+ T cell expansion was observed in the Rx CAR-T c236 and c238 groups in all three models (Figs. 12A-12C).

IFNγ induction was dose dependent in all models (Figs. 13A–13C). Dual constructs at both doses produced 3- to 4-fold more IFNγ at peak in the mixed model than in the KMS11 model alone (Figs. 13A and 13C). Peak induction was observed within 13 days in most groups in all models (peaks at day 20 and day 27 were likely due to GVHD) (Figs. 13A–13C).

In a separate study, day 1 CAR-Ts produced by the GRex 100 and 6-well GRex systems were examined for their anti-tumor activity in vivo using a KMS-11-luc model of disseminated multiple myeloma. The luciferase reporter allows monitoring of disease burden by quantitative bioluminescence imaging (BLI). Briefly, day 1 CAR-Ts manufactured as described above were injected into tumor-bearing mice. Blood samples were collected weekly to measure CAR-T expansion in peripheral blood and analyzed by flow cytometry. T cells engineered using construct #236 and construct #238 demonstrated potent antitumor activity (Fig. 14A) and good CAR-T expansion (Fig. 14B) in vivo in the KMS11 (BCMA+ CD19-) model.

Example 3. Characterization of CAR-T in diabody format

This example describes the characterization of CARs JL1 - JL10 in diabody format. JL1 - JL8 are PI61/CTL119 diabody constructs, and JL9 - JL10 are R1G5/CTL119 diabody constructs. Sequence information for JL1 - JL10 is disclosed in Table 31.

Generation and Measurement of Day 1 BCMA/CD19 Diabody CAR-T Using the Activation Rapid Manufacturing (ARM) Method

In some embodiments, the ARM method was initiated with a frozen or fresh leukapheresis product. After obtaining a sample for counting and QC, the product was connected to a cell sorting device (e.g., a CliniMACS Prodigy device kit) and the program was started. The cells were washed and incubated with microbeads that bind to desired surface markers, such as CD4 and CD8. Bead-labeled cells were selected by passing the cells through a magnetic column. The isolated cells were washed again and the cell media was replaced with separation buffer. The purified T cells were then transferred to culture or cryopreserved for subsequent use. The purity of the isolated T cells was assessed by flow cytometry for a QC step. The cryopreserved cells were thawed, washed in pre-warmed cell media, and resuspended in cell media. Fresh cells were added directly to the culture. Aliquots of frozen total T cell population isolated were thawed in a 37°C water bath, placed in Optimizer CM (Gibco Optimizer medium supplemented with 100 U/ml human IL2) and centrifuged for 5 min at 1500 rpm. Cells were counted and seeded into 24-well plates at 3e6/ml, 1 ml/well. TransAct was added to each well at 1/100 (10 µl/well).

To study the kinetics of CAR expression in vitro, a 24-well plate was used in the ARM assay. At the time of seeding, cells were transduced with a lentiviral vector encoding the CAR for BCMA/CD19 in diabody format at a multiplicity of infection (MOI) of 2. The MOI was determined based on the virus titer obtained in SupT1 cells based on CAR expression in double-positive cells. After 24 h in culture, cells were harvested and washed three times in PBS+1% HSA. Cells were then counted and replated in a 24-well plate at a final number of 1e6/mL. Seventy-two hours after replating, cells were harvested, counted, and a 5e5 cell aliquot from each sample was removed for flow cytometric analysis. This procedure was repeated after 72 h for the day 7 time point.

Figures 15A and 15B show the CAR expression pattern for both BCMA and CD19 96 hours (Figure 15A) or 7 days (Figure 15B) after addition of diabody-encoding virus to primary human T cells made using the ARM method using an MOI of 2. Overall, these results demonstrate that both CARs were well expressed in the diabody format CAR-T system using R1G5 or PI61 in combination with duCD19.1 at day 4 (Figure 15A) or day 7 (Figure 15B) after virus addition. JL1-JL4, JL9, and JL10 showed linear CAR expression in double-positive cells, while JL5-JL8 showed a slight bias toward the CD19+ population, which may be due to differences in CAR binding to the respective detection reagents. Shown here is data from one of two donors.

Producing CAR-T cells using a traditional manufacturing method

The Traditional Manufacturing (TM) method is a method in which T cells are expanded ex vivo for 8-9 days after activation and transduction prior to harvest. Prodigy-treated T cells were resuspended in warm RPMI T cell complete medium (RPMI, 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, 1x NEAA, 1 mM sodium pyruvate, 10 mM HEPES, and 55 μM β-mercaptoethanol) and seeded in 24-well plates at 0.5e ⁶ cells/mL per well. T cells were incubated overnight at 37°C with Human T-Expander CD3/CD28 beads at a bead to cell ratio of 3:1.

On day 1, lentivirus was added at an MOI of 5 based on the titer in SUP-T1. No virus was added to the untransduced control (UTD). T cells were incubated overnight at 37°C followed by the addition of 1 ml of complete T cell medium per well, after which they were incubated overnight at 37°C. For the remaining six days of expansion, the cultures were transferred to tissue culture flasks and diluted with complete T cell medium every two days to achieve a concentration of 0.5e ⁶ cells/ml. Typical dilution ratios ranged from 1:2 to 1:4 throughout the expansion phase.

On day 7, T cells were debeaded and collected and cryopreserved in CryoStor CS10 freezing medium, frozen at -80°C in CoolCell cell freezing containers (BioCision), and transferred to LN ₂ the following day. Small aliquots of T cells were stained for CAR expression. Single-color controls were included for compensation. Samples were measured on a flow cytometer (BD LSRFortessa) and data were analyzed using FlowJo software.

Figure 16 shows CAR expression at day 7 in the TM method using an MOI of 5. The TM products showed an expression pattern similar to that of the ARM products (Figures 15A, 15B, and 16).

In vitro killing assay

The killing potential of T cells engineered with different diabody constructs in response to BCMA- or CD19-expressing target cells was assessed by incubating CAR-T cells with target cells at 2-fold dilutions of E:T ratios starting at 20:1. Target cell numbers were fixed at 2.5 x ¹⁰⁵ cells/well and cells were cultured in 96-well flat-bottomed plates. Effector cells were CAR-T cells generated by conventional diabody-mediated T cell transduction manufacturing. Target cells included BCMA-positive KMS11-luc cells or BCMA-negative NALM6-luc cells. For this assay, % CAR-T cell transduction was normalized by adding UTD to BCMA-CAR-T. This allowed comparisons to be made between the same number of CAR-T and the same total number of T cells in each sample.

The loss of luciferase signal due to cell killing was measured using Bright-Glo substrate 16 hours after cell plating, and specific lysis was calculated using the following formula:

specific lysis (%) = 100 - (sample luminescence/average maximum luminescence) * 100.

Figures 17A and 17B demonstrate the ability of different PI61/CTL119 diabodies to efficiently kill specific target cell lines NALM-6 (CD19+, BCMA-) (Figure 17A) or KMS-11 (CD19-, BCMA+) (Figure 17B). The data suggest that clones JL6, JL5, JL3, and JL8 killed both target cell types equally.

Analysis of cytokine secretion

Supernatants from the co-cultures (1.25:1 ratio) used in the above killing assay were collected after 20 h of incubation for use in the MSD V-plex assay with human IFNγ and IL-2. Different magnitudes of target-specific IFNγ or IL-2 induction were observed by diabody-transduced cells in response to stimulation with KMS11 or NALM6 cells (Fig. 18A and 18B). UTD cells did not show any non-specific IFNγ secretion in response to any target cells (Fig. 18A). Consistent with the killing assay results, clones JL6, JL5, JL3, and JL8 produced more cytokines in response to target cells (Fig. 18A and 18B).

Example 4. Cotransduction of CAR for BCMA and CAR for CD19

Aliquots of frozen total isolated T cells were thawed in a 37°C water bath, placed in Optimizer CM (Gibco Optimizer medium supplemented with 100 U/ml human IL2) and centrifuged for 5 min at 1500 rpm. Cells were counted and seeded into 24-well plates at 3e6/ml, 1 ml/well. TransAct was added to each well at 1/100 (10 μl/well). Virus was added at different multiplicities of infection (MOI) based on SupT1 titer for PI61 or qPCR titer for CTL119. PI61 was used at an MOI of 2 or 1 and paired with three different MOIs for CTL119: 1, 0.5, and 0.25. Mono-CAR was added at an MOI of 1 or 2 for PI61 and 1 for CTL119, and a UTD control was also plated. After 24 h in culture, cells were harvested and washed three times in PBS+1% HSA. Cells were then counted and replated in a 24-well plate at a final density of 1e6/ml.

After 72 hours of replating, cells were harvested, counted, and an aliquot of 5e5 cells from each sample was removed for flow cytometric analysis. Cells were stained with LIVE/DEAD Aqua (BV510) for 15 min at 100 μl/well and then washed twice. Anti-MM antibody (Table 32) was then added at 50 μl/well for 25 min at 4°C. Cells were washed two more times and then fixed for 15 min in 1.6% PFA in PBS, 100 μl/well. After fixation, cells were washed as described previously and resuspended in a final volume of 150 μl/sample in flow cytometry buffer. For each sample, 5e4 cells were gated for live CD3-positive cells on a BD LSRFortessa (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo v.10 software (Ashland, OR). This procedure was repeated 72 hours later for the day 7 time point.

Table 32. Antibody and other reagents Marker Clone Fluorochrome Supplier Catalogue No. Breeding LIVE/
DEAD BV510 Biolegend 423102 1/500 CD3 SK7 BUV395 BD 564001 1/200 CAR19 Anti-idiotypic antibody P.E. Reagent of our own making 1/160 CD4 SK3 PerCP 5.5 Biolegend 344608 1/100 CAR rBCMA-Fc AF 647 Reagent of our own making 1/380 (3 mcg/ml) CD8 SK1 APC H7 BD 560179 1/200 Buffer for FACS Miltenyi Biotec 130-091-222 BSA stock solution Miltenyi Biotec 130-091-376 Phosphate buffered saline (PBS) Gibco 14190-144 Paraformaldehyde (PFA) Polysciences Inc. 18814-10

On day 4 after virus addition, flow cytometry analysis showed significantly higher percentages of the mono-BCMA-CAR+ population compared to the CAR19-positive population and the double-CAR-positive population at an MOI of 2 for BCMA CAR (Figs. 19 and 20). These results were consistent for both donors. With the addition of PI61 at an MOI of 1, good titration of all populations was observed (Figs. 19 and 20). The CAR19+ population as well as the double-CAR+ population decreased as the MOI for CD19 CAR decreased from 1 to 0.25 (Fig. 19). The BCMA-CAR+ population increased with the addition of less virus encoding CTL119 (Fig. 19). The percentages of total CAR+ populations correlated with the total MOI added to each well (Fig. 21). The percentages of total CAR populations for BCMA and CAR19 were also correlated. All trends were stable from day four to day seven (Fig. 21). Viability and expansion rates were not affected by the total MOI added (Fig. 22). Data from one of four donors are shown here.

Example 5. Description of the method of rapid manufacturing with activation (ARM)

In some embodiments, CAR-T cells are manufactured using a continuous rapid activation manufacturing (ARM) process over about 2 days, which will potentially allow for the production of a larger number of less differentiated T cells (naive T cells and T _SCM (central memory T stem cells)) to be returned to the patient for in vivo cell expansion. The short manufacturing time allows T cells with an early differentiation profile to proliferate in the body to their desired final differentiated state, rather than in an ex vivo culture vessel.

In some embodiments, CAR T cells are prepared using cryopreserved leukapheresis-derived starting material, such as unmobilized autologous peripheral blood leukapheresis (LKPK) material. The cryopreserved starting material is subjected to T cell enrichment processing steps on the first day of preparation (day 0) using an immunomagnetic system containing CD4/CD8 antibodies. The positive fraction is then seeded in a GRex culture vessel, activated using an anti-CD3/CD28 antibody system (TransAct), and transduced with a lentiviral vector (LV) encoding a CAR on the same day. The following day, after 20-28 hours of transduction, the T cells are harvested, washed four times, formulated in freezing medium, and then frozen using a programmable cryofreezer (CRF). From the start of the method on day 0 until the start of harvest the following day, cells are cultured for 20-28 hours with a target culture time of 24 hours after seeding on day 0.

Day 0 media were prepared according to Table 21.

Table 21. Media type and point of application during CAR-T manufacturing Medium/buffer type Compound Point of application Buffer for rapid manufacturing method (RB) CliniMACS® Buffer (+ 0.5% Human Serum Albumin (HSA)) Day 0, Cell Washer/Separator Processing Rapid Manufacturing Method (RM) Environment OpTmizer™, CTS™, IL-2, GlutaMAX and ICSR media Day 0 for cell washer/separator and cell seeding processing Collection Buffer (HB) (also called Collection Buffer Solution) PBS without EDTA with 2% HSA Buffer for washing collected material (day 1) Cryopreservation medium Cryostor10 (CS10) Compilation of collected material

The cryopreserved material obtained by leukapheresis is thawed. Thawed cells are diluted with Rapid Method Buffer (Table 21) and washed on a CliniMACS® Prodigy® device. T cells are selected with CliniMACS® anti-CD4 and anti-CD8 beads. After completion of the T cell selection program (approximately 3 hr 40 min to 4 hr 40 min), the reuse bag containing cells suspended in Rapid Method Medium (Table 21) is transferred to a transfer bag. A sample is collected for cell viability and count. Cell count and viability data from the positive fraction in the bag are used to determine the cell concentration for seeding into the culture vessel for activation and vector transduction.

Following positive selection of T cells using CliniMACS® microbeads (with CD4 and CD8 antibodies), cells are seeded into a G-Rex culture vessel. Following cell seeding, activating reagent (TransAct) is then added to the culture vessel. Cells are then transduced with a lentiviral vector encoding CAR at a target MOI of 1.0 (0.8-1.2). Following vector addition, the culture vessel is transported to an incubator where it is incubated for the target time of 24 hours (working range 20-28 hours) at a nominal temperature of 37°C (working range 36-38°C) under nominal 5% _CO2 (working range 4.5-5.5%). Following incubation, the cells are washed with Harvest Wash Solution (Table 21) four times to remove any unintegrated vector and residual viral particles, as well as any other process-related contaminants. The cells are then eluted and a sample is collected for testing to quantify cell numbers and viability, and the results are used to determine the volume required to resuspend the cells for final compilation with the CryoStor® CS10. The cells are then centrifuged to remove Harvest Wash Solution and cryopreserved.

In some embodiments, the CAR expressed in the CAR-T cells binds to CD19. In some embodiments, IL-2 used in the rapid manufacturing (RM) media (Table 21) may be replaced with IL-15, hetIL-15 (IL-15/sIL-15Ra), IL-6, or IL-6/sIL-6Ra.

In some embodiments, the CAR expressed in the CAR-T cells binds to BCMA. In some embodiments, IL-2 used in the rapid manufacturing method (RM) media (Table 21) may be replaced with IL-15, hetIL-15 (IL-15/sIL-15Ra), IL-6, or IL-6/sIL-6Ra.

In some embodiments, the CAR T cells express dual CARs as disclosed herein, such as the dual BCMA/CD19 CARs disclosed herein. In some embodiments, the CAR T cells express a CAR in a diabody format as disclosed herein, such as a CAR in a diabody format as disclosed herein. In some embodiments, the CAR T cells are engineered to express a CAR for BCMA and a CAR for CD19, using cotransduction as disclosed herein.

Example 6: Manufacturing CAR-T cells using the Activated Rapid Manufacturing (ARM) method

The ARM method for producing CAR-T cells begins with the preparation of media as outlined in Table 25.

Cryopreserved leukapheresis product is used as starting material and processed for T cell enrichment. Apheresis documentation, when available, is used to determine the T cell percentage. If T cell percentage data is not available in the apheresis documentation, sentinel vial testing is performed on incoming cryopreserved leukapheresis products to obtain the target T cell percentage for apheresis. The T cell percentage results determine how many bags are thawed on day 0 of the ARM method.

Table 25. Media and buffer type and point of use during CAR-T manufacturing Type of environment Source Point of application CliniMACS® Buffer/Human Serum Albumin (HSA) (0.5% in working concentration) Received by operator on day 0 Day 0, Cell Washer/Separator Processing Environment for the rapid manufacturing method Received by operator on day 0 Day 0 for cell seeding PBS/HSA (1% or 2% in working concentration) Received by operator on day 0 Medium for washing collected and cultured material (day 1) Cryostor10 (CS10) Commercially available Compilation of collected material

The cryopreserved leukapheresis product is thawed, washed, and then subjected to T cell selection and T cell enrichment using CliniMACS® bead technology. Viable nucleated cells (VNCs) are activated using TransAct (Miltenyi) and transduced with a lentiviral vector encoding a CAR. Viable cells selected using Miltenyi beads are plated on a CentriCult in Prodigy®, which is a non-humidified incubation chamber. The cells in culture are suspended in Rapid Process Medium, which is OpTmizer™ CTS™-based medium containing CTS™ Supplement (ThermoFisher), GlutaMAX, IL-2, and 2% Immune Cell Serum Replacer along with its components that promote T cell activation and transduction. Lentiviral transduction is performed once on the day of plating after adding TransAct to diluted cells in the culture medium. The lentiviral vector will be thawed immediately prior to use on the day of plating for up to 30 minutes at room temperature.

From the start of the method on day 0 until the start of the wash and harvest, CAR-T cells are cultured for 20-28 hours after seeding. Following culture, the cell suspension is subjected to two culture washes and one harvest wash in a CentriCult chamber (Miltenyi Biotech).

Following washing of the harvested material on the CliniMACS® Prodigy® on day 1, samples are collected from the cell suspension to determine the number and viability of viable cells. The cell suspension is then transferred to a centrifuge for manual pelleting. The supernatant is discarded and the cell pellet is resuspended in CS10 (BioLife Solution), resulting in a product formulation at a final DMSO concentration of ~10.0%. Viable cell counts are made at the end of harvest for dosing. Doses are then dispensed into individual cryopreservation bags and analytical samples are collected into cryovials.

Cryopreserved products are stored in controlled LN2 storage tanks in a secure area with limited access until final release and transport.

In some embodiments, the CAR T cells express dual CARs as disclosed herein, such as the dual BCMA/CD19 CARs disclosed herein. In some embodiments, the CAR T cells express a CAR in a diabody format as disclosed herein, such as a CAR in a diabody format as disclosed herein. In some embodiments, the CAR T cells are engineered to express a CAR for BCMA and a CAR for CD19, using cotransduction as disclosed herein.

Example 7. Production of CAR T cells expressing CAR for BCMA and CAR for CD19

The rapid CAR T cell production method begins with the preparation of media as outlined in Table 33.

Table 33. Media type and point of application during CAR-T manufacturing Medium/buffer type Compound Stage of the method Rapid Manufacturing Method (RM) Environment OpTmizer CTS
CTS Supplement
ICSR
GlutaMAX
Reconstituted IL-2 Seeding and activation of cells Buffer for rapid manufacturing method (RB) CliniMACS buffer (PBS with EDTA)
HSA Cell washing and T cell enrichment Solution for washing cultured/collected material PBS (without Mg/Ca and EDTA)
HSA Procedure for washing the collected material Cryopreservation medium CryoStor® (CS10) with DMSO Compilation of collected material

The cryopreserved leukapheresis product is thawed. Thawed cells are diluted with Rapid Method Buffer (Table 33) and washed on the CliniMACS® Prodigy® device. T cells are selected using CliniMACS® anti-CD4 and anti-CD8 beads. After completion of the T cell selection program (approximately 3 hr 40 min to 4 hr 40 min), the reuse bag will contain cells suspended in Rapid Method Buffer (Table 33). A sample is collected for cell viability and count. Cell count and viability data from the positive fraction in the bag are used to determine the cell concentration for seeding into the culture vessel for activation and vector transduction.

Following positive selection of T cells using CliniMACS® microbeads (with CD4 and CD8 antibodies), cells are seeded into a CentriCult culture vessel in Prodigy® at a target count of 4.0 x 108 - 1.0 x ¹⁰⁹ total viable cells at a target concentration of approximately 4.0 x ¹⁰⁶ viable cells/mL. After cell seeding, activation reagent (TransAct) is then added to the culture vessel.

The cells are then transduced with a lentiviral vector encoding a CAR for BCMA and a lentiviral vector encoding a CAR for CD19. The volume of vector to be used for transduction of T cells following positive selection is calculated based on a target multiplicity of infection (MOI) of 4.75 for the lentiviral vector encoding a CAR for BCMA and a target MOI of 0.5 for the lentiviral vector encoding a CAR for CD19.

After incubation for the target 24-hour period (working range 20-28 hours) at 37°C under nominal 5% CO2 _, cells are processed to wash the harvested material.

Following incubation, the cells are washed with Harvest Wash Solution (Table 33) three times to remove any unintegrated vector and residual viral particles, as well as any other process-related contaminants. The cells are then eluted and a sample is collected for testing to quantify cell numbers and viability, and the results are used to determine the volume required to resuspend the cells for final compilation with the CryoStor® CS10. The cells are then centrifuged to remove Harvest Wash Solution and cryopreserved.

Example 8. Analysis of gene signatures of CAR-T cells manufactured using the ARM method

Methods

Single cell RNA sequencing

Single-cell RNA-sequencing libraries were generated using the 10X Genomics Chromium Controller and optional library construction kits.

Cryopreserved cells were thawed, counted, and sorted by flow cytometry (if appropriate for the research question) prior to loading onto the 10X Genomics device. Single cells were loaded into droplets, and RNA was barcoded in individual droplets using a GemCode bead. Barcoded RNAs were released from the droplets and processed into an Illumina-compatible whole transcriptome sequencing library.

The generated libraries were sequenced on an Illumina HiSeq and analyzed using the 10X Genomics analysis pipeline and Loupe Cell Browser software.

Single cell immunoprofiling

Whole transcriptome single cell libraries generated by 10X Genomics were used as a reference material for immune cell repertoire profiling and analysis. T cell receptor sequences were PCR amplified from Chromium 5' single cell sequencing libraries and analyzed on an Illumina sequencing machine.

Software pipeline for analysis

Single-cell RNA-seq data were processed using the Cell Ranger analysis pipeline starting from FASTQ files. A detailed description of the Cell Ranger analysis pipeline can be found at support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger. The overall pipeline includes alignment, filtering, barcode scoring, and UMI scoring. Cellular barcodes were used to generate gene-barcode matrices, define clusters, and perform gene expression analysis. Gene expression count data were normalized using Seurat Bioconductor. Cells with fewer than 200 expressed genes were discarded from analysis. Genes that were expressed in only 2 cells or fewer were discarded from analysis. The remaining data were normalized using the Seurat log normalization method using a conversion factor of 10,000. The data were scaled by regression on the number of molecules detected per cell. The gene set score (gene set score) was calculated using the average log-normalized gene expression value for all genes in the gene set. Each gene expression value was normalized using a Z-score such that the mean gene expression value across samples was 0 and the standard deviation was 1. The gene set score was then calculated as the average of the normalized gene expression values in the gene set. An illustrative calculation of the gene set score is described below.

For this example of gene set score calculation, the normalized gene expression values in two (2) samples for six (6) genes are presented in Table 23. For the purposes of this illustrative calculation, the gene set consists of genes 1-4. Therefore, both samples 1 and 2 have a gene set score of 0.

Table 23. Illustrative data set for calculating the gene set score Sample 1 Sample 2 Gen 1 -3 0 Gen 2 3 0 Gen 3 1 0 Gen 4 -1 0 Gen 5 10 4 Gen 6 -5 3

The gene set "upregulated in TEM relative to downregulated in TSCM" includes the following genes: MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RAB27B, ERN1, NPC1, NBEAL2, APOBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PLEKHA5, ADRB2, PLXND1, GNAO1, THBS1, PPP2R2B, CYTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY and GFPT2.

The gene set "upregulated in Tregs relative to downregulated in Teff" includes the following genes: C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DPB2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf53, LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA-DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, CCR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2, LGALS1, ANXA2, PI16, DUSP4, LAYN, ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, LOC541471, RASGRP4, BCAS1, UTS2, MIAT, PRDM1, SEMA3G, FAM129A, HPGD, NCF4, LGALS3, CEACAM4, JAKMIP1, TIGIT, HLA-DRA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIB1, FCRL3 and FOXP3.

The set of genes "repressed in the stemness state" includes the following genes: ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, MEOX1, SFRP1, SP7, SRF, TAL1 and XRCC5.

The set of genes "activated in hypoxia" includes the following genes: ABCB1, ACAT1, ADM, ADORA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA, ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, BNIP3L, BPI, BTG1, C11orf2, C7orf68, CA12, CA9, CALD1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, CITED2, CLK1, CNOT7, COL4A5, COL5A1, COL5A2, COL5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1-Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, EIF4A3, ELF3, ELL2, ENG, ENO1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HMOX1, HMOX2, HSPA5, HSPD1, HSPH1, HYOU1, ICAM1, ID2, IFI27, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IL6, IL8, INSIG1, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LGALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13, MMP2, MMP7, MPI, MT1L, MTL3P, MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2, NOS2P1, NOS2P2, NOS3, NR3C1, NR4A1, NT5E, ODC1, P4HA1, P4HA2, PAICS, PDGFB, PDK3, PFKFB1, PFKFB3, PFKFB4, PFKL, PGAM1, PGF, PGK1, PGK2, PGM1, PIM1, PIM2, PKM2, PLAU, PLAUR, PLIN2, PLOD2, PNN, PNP, POLM, PPARA, PPAT, PROK1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RIOK3, RNASEL, RPL36A, RRP9, SAT1, SERPINB2, SERPINE1, SGSM2, SIAH2, SIN3A, SIRPA, SLC16A1, SLC16A2, SLC20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A16, SLC6A6, SLC6A8, SORL1, SPP1, SRSF6, SSSCA1, STC2, STRA13, SYT7, TBPL1, TCEAL1, TEK, TF, TFF3, TFRC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, THBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC, VIM, VPS11 and XRCC6.

The autophagy-activated gene set includes the following genes: ABL1, ACBD5, ACIN1, ACTRT1, ADAMTS7, AKR1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB, ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, ATG16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, C11orf2, C11orf41, C12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CALCOCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, CISD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRAM2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48A, FANCC, FANCF, FANCL, FBXO7, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP1L, FOXO1, FUNDC1, FUNDC2, FXR2, GABARAP, GABARAPL1, GABARAPL2, GABARAPL3, GABRA5, GDF5, GMIP, HAP1, HAPLN1, HBXIP, HCAR1, HDAC6, HGS, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HK2, HMGB1, HPR, HSF2BP, HSP90AA1, HSPA8, IFI16, IPPK, IRGM, IST1, ITGB4, ITPKC, KCNK3, KCNQ1, KIAA0226, KIAA1324, KRCC1, KRT15, KRT73, LAMP1, LAMP2, LAMTOR1, LAMTOR2, LAMTOR3, LARP1B, LENG9, LGALS8, LIX1, LIX1L, LMCD1, LRRK2, LRSAM1, LSM4, MAP1A, MAP1LC3A, MAP1LC3B, MAP1LC3B2, MAP1LC3C, MAP1S, MAP2K1, MAP3K12, MARK2, MBD5, MDH1, MEX3C, MFN1, MFN2, MLST8, MRPS10, MRPS2, MSTN, MTERFD1, MTMR14, MTMR3, MTOR, MTSS1, MYH11, MYLK, MYOM1, NBR1, NDUFB9, NEFM, NHLRC1, NME2, NPC1, NR2C2, NRBF2, NTHL1, NUP93, OBSCN, OPTN, P2RX5, PACS2, PARK2, PARK7, PDK1, PDK4, PEX13, PEX3, PFKP, PGK2, PHF23, PHYHIP, PI4K2A, PIK3C3, PIK3CA, PIK3CB, PIK3R4, PINK1, PLEKHM1, PLOD2, PNPO, PPARGC1A, PPY, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PRKD2, PRKG1, PSEN1, PTPN22, RAB12, RAB1A, RAB1B, RAB23, RAB24, RAB33B, RAB39, RAB7A, RB1CC1, RBM18, REEP2, REP15, RFWD3, RGS19, RHEB, RIMS3, RNF185, RNF41, RPS27A, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, S100A8, S100A9, SCN1A, SERPINB10, SESN2, SFRP4, SH3GLB1, SIRT2, SLC1A3, SLC1A4, SLC22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, SLC6A1, SLCO1A2, SMURF1, SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, SPATA18, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, STK11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOMM7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VPS33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593 and ZNF681.

The set of genes "upregulated in the resting state relative to downregulated in the activated state" includes the following genes: ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, CD248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, LZTFL1, MANBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISCH, NKTR, NLRP1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1, PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR, PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, PXN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF38, RNF44, ROR1, RPL30, RPL32, RPLP1, RPS20, RPS24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4C, SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG, SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SNPH, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, TECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM66, TMEM8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2, USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBED5, ZBTB25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZFP36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZNF350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862 and ZNF91.

The set of genes "gradually activated during differentiation into memory cells" includes the following genes: MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, C11orf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYO5A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMIP, NAT13, TGFB1, FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3, ADCY9, CD300A, SCD5, ABI3, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, CLIC1, EIF2C4, MAP3K5, IL2RB, PLEKHG1, MYO6, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3, TP53INP1, MBOAT1, GYG1, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1 and KLRB1.

The gene set "upregulated in TEM relative to downregulated in TN" includes the following genes: MYO5A, MXD4, STK3, S1PR5, GLCCI1, CCR3, SOX13, KRTAP5-2, PEA15, PARP8, RNF166, UEVLD, LIMK1, SLC6A6, SV2A, KPNA2, OSBPL7, ST7, GGA2, PI4K2A, CD68, ZAK, RORA, TGFBI, DNAJC1, JOSD1, ZFYVE28, LRP8, OSBPL3, CMIP, NAT13, TGFB1, ANTXR2, NR4A3, RDX, ADCY9, CHN1, CD300A, SCD5, PTPN22, LGALS1, RASGEF1A, GCNT1, GLUL, ABCA2, CLDND1, PAM, CLCF1, MXRA7, CLSTN3, ACOT9, METRNL, BMPR1A, LRIG1, APOBEC3G, CACNB3, RNF19A, RAB27A, FADS3, ACTN4, TBKBP1, FAM53B, MAN1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1, TARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANXA2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21, STOM, PHACTR2, GBP5, ADRB2, PIK3AP1, DUSP10, PTGDR, EOMES, MAF, TPRG1, NBEAL2, NCAPH, SLC4A4, FOSL2, RGS2, TGFBR3, MYO1F, C17orf66, CYTH3, WEE1, PYHIN1, F2R, THBS1, CD58, AUTS2, FAM129A, TNIP3, GZMA, PRR5L, PRDM1, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, ZEB2, CST7, CCL5, GZMK and KLRB1.

Other sets of genes describing similar processes and/or characteristics may also be used to define the characteristics of the cell phenotypes described above.

Cell Ranger VDJ was used to obtain single-cell VDJ sequences and annotations for each single-cell 5' library. Loupe Cell Browser software and Bioconductor packages were used for data analysis and visualization.

Results

The purpose of this example is to compare the T cell status between purified T cells that served as parental cells, CAR-T cells manufactured using the ARM method (referred to as "day 1" cells), and CAR-T cells manufactured using the TM method (referred to as "day 9" cells) using single-cell RNA sequencing (scRNA-seq). In addition, single-cell TCR sequencing (scTCR-seq) was performed to investigate clonality and track cell differentiation from parental material to post-manufacturing material.

As shown in Figs. 23A-23C, naive cells had the lowest gene expression and UMI, suggesting that these cells were not transcriptionally active and were in a quiescent state. Day 1 and day 9 cells expressed more genes, with day 9 cells being the most transcriptionally active. Similar results are shown in Figs. 24A-24D. Naive cells did not express genes related to proliferation (Figs. 24A and 24D).

Additional gene set analysis data are shown in Figs. 25A-25E. Different cell populations were compared using median gene set scores. Day 1 cells and naive cells were in a younger, more memory stem cell-like state (Figs. 25A-25C). In Fig. 25A, the median gene set score values (upregulated in TEMs versus downregulated in TSCMs) for day 1 cells, day 9 cells, and naive cells are -0.084, 0.035, and -0.1, respectively. In Fig. 25B, the median gene set score values (upregulated in Tregs versus downregulated in Teffs) for day 1 cells, day 9 cells, and naive cells are -0.082, 0.087, and -0.071, respectively. In Fig. 25C median gene set score values (silenced in stemness) for day 1 cells, day 9 cells, and parental cells are -0.062, 0.14, and -0.081, respectively.

In addition, day 1 cells were in a more optimal metabolic state compared to day 9 cells (Figs. 25D and 25E). In Fig. 25D, the median gene set scores (hypoxia-activated) for day 1 cells, day 9 cells, and naive cells were 0.019, 0.11, and -0.096, respectively. In Fig. 25E, the median gene set scores (autophagy-activated) for day 1 cells, day 9 cells, and naive cells were 0.066, 0.11, and -0.09, respectively.

The original cells included four clusters based on gene expression. Cluster 0 was characterized by high expression of LMNA, S100A4, etc. Cluster 1 was characterized by high expression of RP913, PRKCQ-AS1, etc. Cluster 2 was characterized by high expression of PR11-291B21.2, CD8B, etc. Cluster 3 was characterized by high expression of NKG7, GZMH, CCL5, CST7, GNLY, FGFBP2, GZMA, CCL4, CTSW, CD8A, etc. In the TSNE plot of the original cells, cluster 3 was separated from other cells, and cluster 1 and cluster 2 were difficult to distinguish.

According to the gene set analysis shown in Fig. 26A-26C, cluster 0 and cluster 3 were enriched in the regulatory T cell phenotype compared to cluster 1 and cluster 2, which were enriched in the effector T cell phenotype. Cluster 3 was dominated by late memory T cells/effector memory T cells (TEM), cluster 1 and cluster 2 were dominated by early memory T cells and naive T cells, and cluster 0 was in the middle. Most of the original cells were in the naive and early memory T cell state. Without being bound by theory, it is believed that such cells may perform best during the manufacturing procedure.

Less transcriptional heterogeneity was observed between day 1 cells and day 9 cells (data not shown).

Like the original population, day 1 cells showed a larger cluster of early memory cells and a smaller cluster of late memory cells in the TSNE plot. This was similar to what was observed with cluster 3 of the original cells. In contrast, day 9 cells did not show distinct clusters of early memory cells in the TSNE plot. This indicates that by day 9 the cells had become more homogeneous.

The TCR was sequenced and clonotype diversity was measured. Overall, the three clonotype profiles were very flat, with most clones occurring only once (Figs. 27A–27C and Table 24). The Shannon entropy in Table 24 is a measure of the flatness of the distribution. The predominant clones in the parental cells were late memory cells. The plot for day 1 cells looked similar to the plot for parental cells but was beginning to level off. By day 9, the plot for the predominant clones had essentially leveled off and the distribution was much flatter. The diversity score was highest on day 9, with day 9 cells having a much more even and flatter distribution than parental or day 1 cells.

Table 24. TCR diversity indices Source cells Product of the day 1 Product of the day 9 Average number of clones per clonotype 1.10 1.05 1.07 Estimated cell count 7344 7687 7233 Total number of clonotypes 5325 7403 6736 Diversity 342.27 802.94 3382,62 Normalized Shannon Entropy 9.98E-01 9,95E-01 9.96E-01

Summary of results

Significant differences in T cell status were observed between day 1 and day 9 products. Day 1 cells were much more similar to parent cells and were enriched in stemness signatures, indicating a more potent product.

Example 9. Cotransduction of CAR for BCMA and CAR for CD19, MOI assessment and efficacy studies

This example describes the characterization of CAR T cells generated by cotransducing T cells with a lentiviral vector encoding a CAR for BCMA and a lentiviral vector encoding a CAR for CD19. The CAR for BCMA comprises the amino acid sequence of SEQ ID NO: 107, and the CAR for CD19 comprises the amino acid sequence of SEQ ID NO: 225.

Cell production and CAR transduction

Four different MOI combinations for BCMA and CD19 CARs in T cell transduction were tested in a 24-well plate format. The lentiviral vector encoding the BCMA CAR and the lentiviral vector encoding the CD19 CAR were used at MOIs of 5 and 1, respectively (5/1 in Fig. 28A and 28B); 5 and 0.5, respectively (5/0.5 in Fig. 28A and 28B); 2.5 and 1, respectively (2.5/1 in Fig. 28A and 28B), or 2.5 and 0.5, respectively (2.5/0.5 in Fig. 28A and 28B). Prodigy purified T cells were seeded at ^3e6 /ml, 1 ml/well in 24-well plates and vectors encoding CARs for both BCMA and CD19 were added at different MOIs as above. After seeding, TransAct polymer nanoarray (Miltenyi Biotec) conjugated with anti-CD3 and anti-CD28 agonist antibodies was added. Cells were incubated in OpTmizer complete T cell medium containing 100 IU/ml recombinant human IL-2 (Prometheus, San Diego, CA) and 2% ICRS (Life Technologies) at 37°C and 5% _CO2 for 24 h prior to harvest.

The cells were then washed three times with 3x PBS+1% HAS. After culturing and washing the collected material, cell viability and number were assessed to determine the effect of vector titration on the final product.

Cells were then re-cultured at 1× ¹⁰⁶ VNC/mL in a 24-well plate and incubated for an additional 6 days. FACS analysis to assess CAR expression for BCMA was performed on days 4 and 7 post-transduction (days 3 and 6 post-harvest).

CAR expression analysis

Samples were measured on a flow cytometer (BD LSRFortessa) and data were analyzed using FlowJo software. In the ARM method, since CAR may not be fully integrated and expressed ex vivo for 24 hours, 96 hours after virus addition can serve as a surrogate time point to determine the in vitro and in vivo dosing strategy with stable CAR expression. The same strategy was used for dual-targeting CAR cocktail measurements.

FACS analyses indicated that cotransduction of T cells with CARs for BCMA and CD19 resulted in three distinct CAR+ subsets, and their relative proportions varied across four different MOI combinations tested (Fig. 28A), indicating that a CAR-T cocktail product could be successfully generated by cotransducing CARs for BCMA and CD19. At an MOI of 5/0.5, the proportion of mono-BCMA-CAR+ (CD19-CAR-negative) was the highest at 20.7%, followed by double-CAR+ at 16.2% and mono-CD19-CAR+ at 10.5%. The total BCMA-CAR+ fraction, calculated as % double CAR+ and % mono-BCMA-CAR+, was 36.9%, and the total CD19-CAR+ fraction was 26.7% (Fig. 28A and 28B). Both the 2.5/0.5 and 2.5/1 MOI ratios resulted in a significantly higher % mono-CD19-CAR+ than % double+ and % mono-BCMA-CAR+. Moreover, % double CAR+ was positively correlated with the total MOI utilization rate (Fig. 28B). The total CAR+ % was similar between the 5/1 MOI and the 5/0.5 MOI. It was observed that CAR expression in each population was relatively stable from day 4 to day 7 post-transduction, including in T cells transduced with CAR for BCMA or CD19 (Fig. 28A and 28B). The data were consistent across the three donors. In some embodiments, an MOI of 4~5 for CAR for BCMA and an MOI of 0.5 for CAR for CD19 were used for co-transduction to produce a dual-targeting CAR-T cocktail.

Additionally, large scale experiments were performed entirely on the CliniMACS Prodigy from T cell enrichment, T cell seeding, activation, transduction and culture to harvest wash in CentriCult prior to formulation and cryopreservation. MOIs of 4/0.5 or 4.75/0.5 were used for BCMA/CD19 CARs. In this study, a seeding density of ^3e6 /mL was used in a total volume of 250 mL to yield a total of ^7.5e8 cells. Cells were harvested after 24 h, washed 3 times with PBS+1% HAS and cryopreserved for subsequent use. The remaining T cells were pooled to generate the respective UTD, BCMA-CAR-T and CD19-CAR-T controls. An aliquot was removed and recultured at ^1e6 /ml in a 24-well plate for flow cytometric analysis to assess BCMA-CAR expression at day 4 post-transduction.

Figure 29 shows that the dual-targeting CAR-T cocktail contained 11.8% mono-BCMA CAR T cells, 4.31% dual-specificity CAR T cells, and ~9% mono-CD19 CAR T cells at day 4 post-transduction. In this study, the total BCMA-CAR+ %, calculated as the dual-CAR+ % plus the mono-BCMA-CAR+ %, was 16%; and the total CD19-CAR+ %, calculated as the dual-CAR+ % plus the mono-CD19-CAR+ %, was 13.4%.

The in vivo activity of a dual-targeting CAR-T cocktail was analyzed in three xenograft mouse models: 1) a KMS-11-luc multiple myeloma model (this BCMA-expressing model is labeled with a luciferase reporter construct, allowing the disease to be monitored systemically in the bone marrow via bioluminescent imaging); 2) a mixed tumor model created by mixing 95% KMS-11-luc myeloma cells with 5% CD19+ tumor cells (e.g., Nalm-6-luc) to mimic the heterogeneity in MM patients; and 3) a Nalm-6-luc model to assess the specificity of CD19 targeting and further expand the double-positive CAR-T population in the dual-targeting CAR-T cocktail. BCMA-CAR-T and CD19-CAR-T served as controls for their respective models. To calculate the CAR-T cell dose for models 1 and 2, total BCMA-CAR+% was measured at day 4 post-transduction, while for model 3, total CD19-CAR+% was measured at day 4 post-transduction. The UTD dose reflected the highest dose of the total T cell population in the respective method.

Tumor regression curves for all groups in the three models are shown in Figs. 30A-E. In the KMS-11-luc model, both the dual-targeting CAR-T cocktail and BCMA-CAR-T induced tumor regression in a dose-dependent manner, as shown in Fig. 30A. At the 1e ⁴ dose, both the dual-targeting CAR-T cocktail and BCMA-CAR-T demonstrated delayed tumor inhibition at a similar rate, and both were able to achieve tumor clearance at the end of the study. The dual-targeting CAR-T cocktail demonstrated more effective tumor clearance than BCMA-CAR-T at the 5e ⁴ dose, with the dual-targeting CAR-T cocktail achieving tumor clearance by day 14 after CAR-T infusion, whereas BCMA-CAR-T demonstrated an effect delayed by at least one week. In a repeat study using the remaining cell products from the same lot and covering wider dose ranges ( ^1e4 , ^5e4 , ^1.5e5 ) (Fig. 30B), the dose response was confirmed for tumor regression with the dual-targeting CAR-T cocktail. The regression curves for the dual-targeting CAR-T cocktail at ^1.5e5 , ^5e4 and for BCMA-CAR-T at ^1.5e5 are superimposed. BCMA-CAR-T at ^5e4 was able to eradicate tumors by day 14 post-CAR-T infusion in this cohort of mice, despite slightly slower tumor regression than the dual-targeting CAR-T cocktail at the same dose through 14 days post-CAR-T infusion. At dose 1e ^4, the dual-targeting CAR-T cocktail demonstrated superior efficacy to BCMA-CAR-T. Briefly, using the KMS-11-luc model, it was demonstrated that the dual-targeting CAR-T cocktail was able to specifically target BCMA, was as active as BCMA-CAR-T in killing BCMA+ multiple myeloma tumor cells, and was even more active than BCMA-CAR-T at lower doses.

Next, the efficacy of the dual-targeting CAR-T cocktail was assessed in the presence of both BCMA and CD19 in a “mixed” model in which KMS-11-luc cells mixed with 5% Nalm-6-luc cells were implanted in mice. In the mixed tumor model, as shown in Fig. 30C, only the dual-targeting CAR-T cocktail at ^5e4 and ^1e4 showed partial tumor inhibition in a dose-dependent manner, whereas neither BCMA-CAR-T nor CD19-CAR-T showed any effect at the ^5e4 dose. In a replication study involving a higher dose group, as shown in Fig. 30D, it was demonstrated that the dual-targeting CAR-T cocktail at ^1.5e5 was able to eradicate mixed tumors by day 14 post-CAR-T infusion, and the tumor suppression was dose-dependent. In contrast, BCMA-CAR-T and CD19-CAR-T at 1.5e ⁵ showed only partial tumor inhibition in this mixed model in the presence of both BCMA and CD19.

Finally, the Nalm-6-luc model was used to show whether the dual-targeting CAR-T cocktail could specifically target CD19+ tumors. As shown in Fig. 30E, the dual-targeting CAR-T cocktail was also able to eliminate Nalm-6-luc tumors in a dose-dependent manner that was comparable to mono-CD19-CAR-T at the higher dose and was better than mono-CD19-CAR-T at the lower dose.

Example 10. MOI evaluation for manufacturing a BCMA/CD19-CAR-T cell product

Based on the titer determined by qPCR, titration of BCMA CAR-encoding virus was performed to determine the optimal BCMA CAR/CD19 CAR vector ratio. Briefly, T cells were thawed and resuspended at a density of 3 x ¹⁰⁶ VNC/mL or 6 x ¹⁰⁶ VNC/mL. For each MOI tested, 1 ml of the cell suspension was seeded into a 24-well plate, transduced at time 0, and incubated for 20-24 h. Then, 1 ml of each culture was manually washed with 3 x 2 ml PBS + 1% HSA. After culturing and washing the harvested material, cell viability and number were assessed to determine the impact of vector titration on the final product. The collected cells were then placed in culture at 1×10 ⁶ VNC/mL and CAR expression (for BCMA and for CD19) was measured at 72 h and 144 h post-collection (day 3 and day 6 post-collection (PH) (day 4 and day 7 post-transduction), Table 34 and Fig. 31).

Table 34. GMP-compliant vector titration for transduction to produce a BCMA/CD19 dual-specificity CAR-T cell product Donor ID Day 0 seeding density (VNC/ml) MOI (TE/cell),
CAR for BCMA/CAR for CD19 % CAR (total population with CAR for BCMA = population with mono-BCMA-CAR + population with dual CAR for BCMA/CD19)
Day 3 PH Day 6 PH % mono-CD19-CAR
Day 3 PH
Day 6 PH Donor A 3e6 4/0.5 34.4 40.6 11.0 14.4 5/0.5 38.3 49.1 8.9 12.1 6e6 4/0.5 32.6 40.7 9.4 13.3 5/0.5 38.1 47.8 8.7 11.7 Donor B 3e6 4/0.5 38.0 47.9 11.1 14.9 5/0.5 40.6 52.9 9.7 12.6 6e6 4/0.5 40.6 51.2 12.1 16.7 5/0.5 43.0 55.9 9.9 13.6

In some embodiments, the total number of viable BCMA-CAR+ T cells measured by flow cytometry at day 4 post-transduction (or day 3 post-harvest) is used for dose calculations. In some embodiments, the T cells are seeded at a density of about 4e6 VNC/mL in CentriCult. In some embodiments, the vector encoding the CAR for BCMA is used at an MOI of 4.75, and the vector encoding the CAR for CD19 is used at an MOI of 0.5.

In a separate study, BCMA/CD19 CAR T cells were manufactured using the rapid co-transduction manufacturing approach described above at scale in a CliniMACS Prodigy automated closed system device. Briefly, the process begins with the selection of T cells from cryopreserved buffy coated cells. T cells are positively selected with anti-CD4 and anti-CD8 microbeads. Following selection, T cells are eluted into a reuse bag and a sample is collected for assessment of cell concentration, viability, and purity. Cells are then activated and transduced with a lentiviral vector encoding a CAR for BCMA and a lentiviral vector encoding a CAR for CD19. As shown in Fig. 32, the T cell subsets in the Prodigy output were indistinguishable from the apheresis input.

Example 11. Further characterization of T cells engineered to express CAR for BCMA and CAR for CD19

This example describes further characterization of a BCMA/CD19-CAR-T cell product that was produced as described in Example 9. This cell product contains three different CAR+ populations: mono-BCMA-CAR-T cells, mono-CD19-CAR-T cells, and double-positive BCMA/CD19-CAR-T cells. In addition, this cell product also contains a population of untransduced T cells.

IFN γ in plasma of mice treated with a BCMA/CD19 dual-targeting CAR-T cell product

IFNγ is a hallmark of CAR-T cell activation in response to target binding. Plasma IFNγ kinetics were analyzed in the in vivo studies described in Example 9. As shown in Figs. 33A and 33B, all CAR-T-treated groups demonstrated low levels of circulating IFNγ (10-50 pg/mL) at day 2, followed by a continued increase in IFNγ secretion.

Pharmacokinetic parameters and metabolism in non-clinical studies

Peripheral blood expansion of the BCMA/CD19 dual-specific CAR-T cell product (comprising mono-BCMA, mono-CD19, and double-positive BCMA/CD19 CAR-T cells) was analyzed by flow cytometry for up to 3 weeks post-infusion and compared to the reference BCMA CAR-T in the KMS-11-luc model. Both CD3+ T cell and CAR+ T cell expansion were observed in all CAR-T treatment groups. Dose-dependent cell expansion was observed for the BCMA/CD19 dual-specific CAR-T cell product in the double-positive or mono-CAR+ T cell population. Based on data from two studies, the BCMA/CD19 dual-specificity CAR-T cell product demonstrated slightly greater expansion of BCMA-targeting CAR+ T cells and the total CAR+ T cell population compared with BCMA-CAR-T.

Antigen-dependent expansion of the BCMA/CD19 dual-specificity CAR-T cell product was demonstrated by assessing CAR+ T cell expansion in three models (KMS-11, Nalm-6, and mixed). In the BCMA-expressing KMS-11 xenograft model, BCMA/CD19 double-positive and mono-BCMA-CAR+ T cells were extensively expanded, whereas in the CD19-expressing Nalm-6 xenograft model, BCMA/CD19 double-positive and mono-CD19-CAR+ T cells were extensively expanded. The double-positive CAR-T cell population was able to expand upon activation with either BCMA or CD19 antigen alone. The initial expansion rates of double-positive BCMA/CD19- and mono-BCMA-CAR+ T cells were comparable in the KMS-11 model, as were the expansion rates of double-CAR-positive and mono-CD19-CAR+ T cells in the Nalm-6 model.

Conclusions

The BCMA/CD19 Dual Specificity CAR-T Cell Product is a novel CAR-T cell product with dual targeting of BCMA and CD19, produced using a rapid manufacturing process that preserves T cell stemness.

In three xenograft mouse models (KMS-11, Nalm-6 and mixed), the dual-specificity BCMA/CD19 CAR-T cell product demonstrated potent in vivo pharmacological properties by controlling tumor growth, inducing CAR-T expansion and cytokine production in an antigen-dependent and dose-dependent manner.

Tumor eradication in the mixed model was achieved with the BCMA/CD19 dual-targeting CAR-T cell product, with tumor regression increasing in a dose-dependent manner. Neither mono-BCMA-CAR-T nor mono-CD19-CAR-T demonstrated tumor regression in the mixed model. Furthermore, the BCMA/CD19 dual-targeting CAR-T cell product demonstrated extensive CAR-T expansion in vivo , whereas the dual-positive CAR-T population expanded when activated by either BCMA or CD19 alone. In the KMS-11-luc multiple myeloma model, the BCMA/CD19 dual-targeting CAR-T cell product demonstrated improved tumor growth control at higher dose levels tested and better tumor growth control at lower dose levels tested compared to BCMA-CAR-T.

These results confirm that the BCMA/CD19 dual-specificity CAR-T cell product is a dual-targeting CAR-T that has the potential to transform clinical outcomes by addressing the potential contribution of BCMA-/CD19+ stem/progenitor cells to multiple myeloma relapse, potentially providing deeper and more durable responses than conventionally manufactured CAR-Ts or those targeting a single BCMA antigen.

Example 12. Phase I clinical trial of BCMA CAR T cells manufactured using the ARM method

This example describes an open-label, phase I study to evaluate the safety and tolerability of a BCMA CAR T cell-based therapy manufactured using the ARM process in adult patients with relapsed and/or refractory multiple myeloma.

Primary outcome measures include the incidence and nature of dose-limiting toxicities (DLTs) within the first 28 days after BCMA-CAR-T cell administration, and the incidence and severity of adverse events (AEs) and serious adverse events (SAEs), including laboratory, ECG, and vital sign changes after BCMA-CAR-T cell administration.

Secondary outcome measures include manufacturing success rate (defined as the number of subjects treated at the planned target dose divided by the total number of subjects treated), ORR (the proportion of subjects with best overall response (BOR) of sCR+CR+VGPR+PR at months 3 and 6 as determined by the local investigator using the IMWG criteria (Kumar et al., Lancet Oncol. 2016 Aug;17(8):e328–e346, incorporated herein by reference in its entirety)), CRR (the proportion of subjects with BOR of sCR+CR at month 3 as determined by the local investigator using the IMWG criteria), local investigator-assessed DOR (time from achieving sCR+CR+VGPR+PR to MM-related relapse or death), qPCR-detectable CAR-T transgene concentrations over time in peripheral blood and bone marrow, and a summary of pre-existing and treatment-induced immunogenicity (cellular and humoral) of BCMA-CAR-T cell-based therapy.

The inclusion criteria are as follows:

subjects with MM who have relapsed and/or are refractory to at least 2 prior treatment regimens, including an IMiD (eg, lenalidomide or pomalidomide), a proteasome inhibitor (eg, bortezomib, carfilzomib), and an approved anti-CD38 antibody (eg, daratumumab), if available, and who have documented evidence of disease progression (per IMWG criteria);

measurable disease defined by the protocol;

ECOG performance status score of 0 or 1 at the time of screening;

proper hematological parameters and

the presence of material obtained by leukapheresis from unmobilized cells, accepted for manufacturing.

The exclusion criteria are as follows:

prior administration of a genetically modified cell product, including prior BCMA-CAR-T-based therapy. Patients previously treated with bispecific antibodies or antibody-drug conjugates (ADCs) targeting BCMA are not excluded;

autologous HSCT within 6 weeks prior to study inclusion or any previous allogeneic hematopoietic stem cell transplant (HSCT);

chemotherapy or any concomitant anticancer therapy (except protocol-prescribed chemotherapy with lymphodepletion (LD)) within 2 weeks prior to apheresis;

treatment with small molecule targeted antineoplastic agents for 2 weeks prior to apheresis product collection or 5 half-lives, whichever is shorter; and

receipt of antibodies or immunotherapeutic agents (except daratumumab) within 4 weeks prior to apheresis collection. Daratumumab within 3 weeks prior to apheresis collection.

Example 13. Evaluation of BCMA/CD19-CAR-T in diabody format

The efficacy of the CD19 antigen response element in the novel single-chain diabody CAR T cells is assessed using the NALM-6 luciferase model. 1 x 10 ⁶ NALM-6-Luc cells are implanted by injection into the lateral tail vein on day -7 of CAR-T dosing. Body weight is measured and in vivo bioluminescence imaging (BLI) is performed twice weekly to assess tumor progression. Animals are measured twice weekly and randomized to a specific group (day -1) after achieving a tumor burden corresponding to a photon flux of 3 x 10 ⁶ (photons/second). On day 0, diabody CAR T cells are removed from liquid nitrogen and thawed for injection. 1 x 10 ⁶ doubly positive CAR cells are injected into the lateral tail vein. An experimental evaluation is conducted over several weeks to determine which design has the best functional effectiveness by assessing the decrease in BLI intensity over time.

EQUIVALENTS

The disclosures of any and all patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that additional embodiments and modifications of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed as including all such embodiments and equivalent modifications.

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SEQUENCE LIST

<110> NOVARTIS AG

<120> CHIMERIC ANTIGEN RECEPTORS AND WAYS OF THEIR APPLICATION

<130> N2067-7166WO

<140>

<141>

<150> 62/940,509

<151> 2019-11-26

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<170> PatentIn version 3.5

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His Ala Ala Arg Pro

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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

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Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

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Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

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Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

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Leu Ser Leu Gly Lys Met

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Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

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Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

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Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

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Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

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Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

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Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

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Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

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Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

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Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

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Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

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Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

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Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

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Ser Leu Glu Val Ser Tyr Val Thr Asp His

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<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

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cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tggggggagaa ccgtatataa 180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360

ggtggggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840

ggagaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140

agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184

<210> 12

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

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<400> 12

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ccc 63

<210> 13

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

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<400> 13

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 14

<211> 690

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

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<400> 14

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaagatg 690

<210> 15

<211> 847

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 15

aggtggcccg aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca 60

gaaggcagcc tagccaaagc tactactgca cctgccacta cgcgcaatac tggccgtggc 120

ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga gaccaagacc 180

cctgaatgtc catcccatac ccagccgctg ggcgtctatc tcttgactcc cgcagtacag 240

gacttgtggc ttagagataa ggccaccttt acatgtttcg tcgtgggctc tgacctgaag 300

gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg 360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga 420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca 480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat 540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc 600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc 660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt 720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc 780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact 840

gaccatt 847

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 16

ggtggcggag gttctggagg tggaggttcc 30

<210> 17

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 17

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 18

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 18

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 19

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 19

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 20

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 20

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 21

<211> 336

<212> DNA

<213> Homo sapiens

<400> 21

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 22

<211> 373

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 22

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 23

<211> 1182

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 23

atggccctcc ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga 60

ccacccggat ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg 120

gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt ctccaacacc 180

tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt caaaccagac cgacaagctc 240

gccgcgtttc cggaagatcg gtcgcaaccg ggacaggatt gtcggttccg cgtgactcaa 300

ctgccgaatg gcagagactt ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg 360

acctacctgt gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg 420

gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc atccccatcg 480

cctcggcctg cggggcagtt tcagaccctg gtcacgacca ctccggcgcc gcgcccaccg 540

actccggccc caactatcgc gagccagccc ctgtcgctga ggccggaagc atgccgccct 600

gccgccggag gtgctgtgca tacccgggga ttggacttcg catgcgacat ctacatttgg 660

gctcctctcg ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc 720

aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag gcccgtgcaa 780

accacccagg aggaggacgg ttgctcctgc cggttccccg aagaggaaga aggaggttgc 840

gagctgcgcg tgaagttctc ccggagcgcc gacgcccccg cctataagca gggccagaac 900

cagctgtaca acgaactgaa cctgggacgg cgggaagagt acgatgtgct ggacaagcgg 960

cgcggccggg accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg 1020

tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg gatgaaggga 1080

gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag gactgtccac cgccaccaag 1140

gacacatacg atgccctgca catgcaggcc cttccccctc gc 1182

<210> 24

<211> 394

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 24

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

20 25 30

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

35 40 45

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

50 55 60

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

65 70 75 80

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

85 90 95

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

100 105 110

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

115 120 125

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

130 135 140

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

145 150 155 160

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

165 170 175

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

180 185 190

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

195 200 205

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

210 215 220

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

225 230 235 240

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

245 250 255

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

260 265 270

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

275 280 285

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

290 295 300

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

305 310 315 320

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

325 330 335

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

340 345 350

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

355 360 365

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<210> 25

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 25

Gly Gly Gly Gly Ser

1 5

<210> 26

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<220>

<221> SITE

<222> (1)..(30)

<223> /note="This sequence may encompass 1-6 'Gly Gly Gly Gly Ser'

repeating units"

<220>

<221> source

<223> /note="See specification as filed for detailed description of

substitutions and preferred embodiments"

<400> 26

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 27

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 27

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 28

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 28

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 29

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 29

Gly Gly Gly Ser

1

<210> 30

<211> 5000

<212> RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<220>

<221> misc_feature

<222> (1)..(5000)

<223> /note="This sequence may encompass 50-5000 nucleotides"

<400> 30

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980

aaaaaaaaaa aaaaaaaaaa 5000

<210> 31

<211> 100

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 31

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 60

tttttttttt tttttttttt tttttttttt tttttttttt 100

<210> 32

<211> 5000

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<220>

<221> misc_feature

<222> (1)..(5000)

<223> /note="This sequence may encompass 50-5000 nucleotides"

<400> 32

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 60

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 1920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 1980

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2040

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2100

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2160

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2220

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2280

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2340

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2400

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2460

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2520

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2580

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2640

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2700

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2760

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2820

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2880

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 2940

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3000

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3060

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 3960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt 4980

tttttttttt tttttttttt 5000

<210> 33

<211> 5000

<212> RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<220>

<221> misc_feature

<222> (1)..(5000)

<223> /note="This sequence may encompass 100-5000 nucleotides"

<400> 33

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4980

aaaaaaaaaa aaaaaaaaaa 5000

<210> 34

<211> 400

<212> RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<220>

<221> misc_feature

<222> (1)..(400)

<223> /note="This sequence may encompass 100-400 nucleotides"

<400> 34

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 400

<210> 35

<211> 2000

<212> RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<220>

<221> misc_feature

<222> (1)..(2000)

<223> /note="This sequence may encompass 50-2000 nucleotides"

<400> 35

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaaaaaaa aaaaaaaaaa 2000

<210> 36

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 36

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 37

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 37

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 38

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 38

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210> 39

<211> 105

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 39

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta 105

<210> 40

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 40

ggtggcggag gttctggagg tggggggttcc 30

<210> 41

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 41

Gly Gly Gly Ser

1

<210> 42

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<220>

<221> SITE

<222> (1)..(40)

<223> /note="This sequence may encompass 1-10 'Gly Gly Gly Ser'

repeating units"

<400> 42

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser

35 40

<210> 43

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 43

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 44

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 44

Ser Tyr Ala Met Ser

1 5

<210> 45

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 45

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 46

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 46

Arg Glu Trp Val Pro Tyr Asp Val Ser Trp Tyr Phe Asp Tyr

1 5 10

<210> 47

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 47

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 48

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 48

Ser Gly Ser Gly Gly Ser

1 5

<210> 49

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 49

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 50

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 50

Ile Ser Gly Ser Gly Gly Ser Thr

1 5

<210> 51

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 51

Ala Arg Arg Glu Trp Val Pro Tyr Asp Val Ser Trp Tyr Phe Asp Tyr

1 5 10 15

<210> 52

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 52

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Val Pro Tyr Asp Val Ser Trp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 53

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tgggtgccct acgatgtcag ctggtacttc gactactggg gacagggcac tctcgtgact 360

gtgtcctcc 369

<210> 54

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 54

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 55

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 55

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 56

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 56

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210> 57

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 57

Ser Gln Ser Ile Ser Ser Tyr

1 5

<210> 58

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 58

Ala Ala Ser

1

<210> 59

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 59

Ser Tyr Ser Thr Pro Leu

1 5

<210> 60

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 60

Gln Ser Ile Ser Ser Tyr

1 5

<210> 61

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 62

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 62

gacattcaaa tgactcagtc cccgtcctcc ctctccgcct ccgtgggaga tcgcgtcacg 60

atcacgtgca gggccagcca gagcatctcc agctacctga actggtacca gcagaagcca 120

gggaaggcac cgaagctcct gatctacgcc gctagctcgc tgcagtccgg cgtcccttca 180

cggttctcgg gatcgggctc aggcaccgac ttcaccctga ccattagcag cctgcagccg 240

gaggacttcg cgacatacta ctgtcagcag tcatactcca cccctctgac cttcggccaa 300

gggaccaaag tggagatcaa g 321

<210> 63

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 63

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 64

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 64

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Val Pro Tyr Asp Val Ser Trp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

145 150 155 160

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu

165 170 175

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

180 185 190

Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

210 215 220

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr

225 230 235 240

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

245 250

<210> 65

<211> 750

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 65

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tgggtgccct acgatgtcag ctggtacttc gactactggg gacagggcac tctcgtgact 360

gtgtcctccg gtggtggtgg atcggggggt ggtggttcgg gcggaggagg atctggagga 420

ggagggtcgg acattcaaat gactcagtcc ccgtcctccc tctccgcctc cgtggggagat 480

cgcgtcacga tcacgtgcag ggccagccag agcatctcca gctacctgaa ctggtaccag 540

cagaagccag ggaaggcacc gaagctcctg atctacgccg ctagctcgct gcagtccggc 600

gtcccttcac ggttctcggg atcgggctca ggcaccgact tcaccctgac cattagcagc 660

ctgcagccgg aggacttcgc gacatactac tgtcagcagt catactccac ccctctgacc 720

ttcggccaag ggaccaaagt ggagatcaag 750

<210> 66

<211> 473

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 66

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Val Pro Tyr Asp Val Ser Trp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

145 150 155 160

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu

165 170 175

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

180 185 190

Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

210 215 220

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr

225 230 235 240

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro

245 250 255

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

260 265 270

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

275 280 285

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

290 295 300

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

305 310 315 320

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

325 330 335

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

340 345 350

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

355 360 365

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

370 375 380

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

385 390 395 400

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

405 410 415

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

420 425 430

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

435 440 445

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

450 455 460

Leu His Met Gln Ala Leu Pro Pro Arg

465 470

<210> 67

<211> 1419

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 67

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tgggtgccct acgatgtcag ctggtacttc gactactggg gacagggcac tctcgtgact 360

gtgtcctccg gtggtggtgg atcggggggt ggtggttcgg gcggaggagg atctggagga 420

ggagggtcgg acattcaaat gactcagtcc ccgtcctccc tctccgcctc cgtggggagat 480

cgcgtcacga tcacgtgcag ggccagccag agcatctcca gctacctgaa ctggtaccag 540

cagaagccag ggaaggcacc gaagctcctg atctacgccg ctagctcgct gcagtccggc 600

gtcccttcac ggttctcggg atcgggctca ggcaccgact tcaccctgac cattagcagc 660

ctgcagccgg aggacttcgc gacatactac tgtcagcagt catactccac ccctctgacc 720

ttcggccaag ggaccaagt ggagatcaag accactaccc cagcaccgag gccacccacc 780

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 840

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 900

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 960

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1020

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1080

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1140

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1200

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1260

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1320

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1380

acctatgacg ctcttcacat gcaggccctg ccgcctcgg 1419

<210> 68

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 68

Arg Glu Trp Trp Tyr Asp Asp Trp Tyr Leu Asp Tyr

1 5 10

<210> 69

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 69

Ala Arg Arg Glu Trp Trp Tyr Asp Asp Trp Tyr Leu Asp Tyr

1 5 10

<210> 70

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 70

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Tyr Asp Asp Trp Tyr Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 71

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 71

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tggtggtacg acgattggta cctggactac tggggacagg gcactctcgt gactgtgtcc 360

tcc 363

<210> 72

<211> 248

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 72

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Tyr Asp Asp Trp Tyr Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys

245

<210> 73

<211> 744

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 73

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tggtggtacg acgattggta cctggactac tggggacagg gcactctcgt gactgtgtcc 360

tccggtggtg gtggatcggg gggtggtggt tcgggcggag gaggatctgg aggagggaggg 420

tcggacattc aaatgactca gtccccgtcc tccctctccg cctccgtggg agatcgcgtc 480

acgatcacgt gcagggccag ccagagcatc tccagctacc tgaactggta ccagcagaag 540

ccagggaagg caccgaagct cctgatctac gccgctagct cgctgcagtc cggcgtccct 600

tcacggttct cgggatcggg ctcaggcacc gacttcaccc tgaccattag cagcctgcag 660

ccggaggact tcgcgacata ctactgtcag cagtcatact ccacccctct gaccttcggc 720

caagggacca aagtggagat caag 744

<210> 74

<211> 471

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 74

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Tyr Asp Asp Trp Tyr Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 75

<211> 1413

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 75

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tggtggtacg acgattggta cctggactac tggggacagg gcactctcgt gactgtgtcc 360

tccggtggtg gtggatcggg gggtggtggt tcgggcggag gaggatctgg aggagggaggg 420

tcggacattc aaatgactca gtccccgtcc tccctctccg cctccgtggg agatcgcgtc 480

acgatcacgt gcagggccag ccagagcatc tccagctacc tgaactggta ccagcagaag 540

ccagggaagg caccgaagct cctgatctac gccgctagct cgctgcagtc cggcgtccct 600

tcacggttct cgggatcggg ctcaggcacc gacttcaccc tgaccattag cagcctgcag 660

ccggaggact tcgcgacata ctactgtcag cagtcatact ccacccctct gaccttcggc 720

caagggacca aagtggagat caagaccact accccagcac cgaggccacc caccccggct 780

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt 840

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg 900

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt 960

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 1020

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc 1080

gtgaaattca gccgcagcgc agatgctcca gcctaccagc aggggcagaa ccagctctac 1140

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1200

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag 1260

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga 1320

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat 1380

gacgctcttc acatgcaggc cctgccgcct cgg 1413

<210> 76

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 76

Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr

1 5 10

<210> 77

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 77

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr

1 5 10

<210> 78

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 78

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 79

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 79

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tggtggggag aaagctggct gttcgactac tggggacagg gcactctcgt gactgtgtcc 360

tcc 363

<210> 80

<211> 248

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 80

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys

245

<210> 81

<211> 744

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 81

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tggtggggag aaagctggct gttcgactac tggggacagg gcactctcgt gactgtgtcc 360

tccggtggtg gtggatcggg gggtggtggt tcgggcggag gaggatctgg aggagggaggg 420

tcggacattc aaatgactca gtccccgtcc tccctctccg cctccgtggg agatcgcgtc 480

acgatcacgt gcagggccag ccagagcatc tccagctacc tgaactggta ccagcagaag 540

ccagggaagg caccgaagct cctgatctac gccgctagct cgctgcagtc cggcgtccct 600

tcacggttct cgggatcggg ctcaggcacc gacttcaccc tgaccattag cagcctgcag 660

ccggaggact tcgcgacata ctactgtcag cagtcatact ccacccctct gaccttcggc 720

caagggacca aagtggagat caag 744

<210> 82

<211> 471

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 82

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 83

<211> 1413

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 83

gaagtgcagt tgctggagtc aggcggagga ctggtgcagc ccggaggatc gcttcgcttg 60

agctgcgcag cctcaggctt taccttctcc tcctacgcca tgtcctgggt cagacaggct 120

cccgggaagg gactggaatg ggtgtccgcc attagcggtt ccggcggaag cacttactat 180

gccgactctg tgaagggccg cttcactatc tcccgggaca actccaagaa caccctgtat 240

ctccaaatga attccctgag ggccgaagat accgcggtgt actactgcgc tagacgggag 300

tggtggggag aaagctggct gttcgactac tggggacagg gcactctcgt gactgtgtcc 360

tccggtggtg gtggatcggg gggtggtggt tcgggcggag gaggatctgg aggagggaggg 420

tcggacattc aaatgactca gtccccgtcc tccctctccg cctccgtggg agatcgcgtc 480

acgatcacgt gcagggccag ccagagcatc tccagctacc tgaactggta ccagcagaag 540

ccagggaagg caccgaagct cctgatctac gccgctagct cgctgcagtc cggcgtccct 600

tcacggttct cgggatcggg ctcaggcacc gacttcaccc tgaccattag cagcctgcag 660

ccggaggact tcgcgacata ctactgtcag cagtcatact ccacccctct gaccttcggc 720

caagggacca aagtggagat caagaccact accccagcac cgaggccacc caccccggct 780

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt 840

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg 900

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt 960

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 1020

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc 1080

gtgaaattca gccgcagcgc agatgctcca gcctaccagc aggggcagaa ccagctctac 1140

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg 1200

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag 1260

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga 1320

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat 1380

gacgctcttc acatgcaggc cctgccgcct cgg 1413

<210> 84

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace=" "

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace=" "

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="Y"

<220>

<221> VARIANT

<222> (7)..(7)

<223> /replace="Y" or "D"

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace="D" or "V"

<220>

<221> VARIANT

<222> (9)..(9)

<223> /replace="D"

<220>

<221> VARIANT

<222> (11)..(11)

<223> /replace="Y"

<220>

<221> VARIANT

<222> (12)..(12)

<223> /replace="L"

<220>

<221> SITE

<222> (1)..(14)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 84

Arg Glu Trp Val Pro Trp Gly Glu Ser Trp Leu Phe Asp Tyr

1 5 10

<210> 85

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace=" "

<220>

<221> VARIANT

<222> (7)..(7)

<223> /replace=" "

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace="Y"

<220>

<221> VARIANT

<222> (9)..(9)

<223> /replace="Y" or "D"

<220>

<221> VARIANT

<222> (10)..(10)

<223> /replace="D" or "V"

<220>

<221> VARIANT

<222> (11)..(11)

<223> /replace="D"

<220>

<221> VARIANT

<222> (13)..(13)

<223> /replace="Y"

<220>

<221> VARIANT

<222> (14)..(14)

<223> /replace="L"

<220>

<221> SITE

<222> (1)..(16)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 85

Ala Arg Arg Glu Trp Val Pro Trp Gly Glu Ser Trp Leu Phe Asp Tyr

1 5 10 15

<210> 86

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 86

Ser Tyr Gly Met His

1 5

<210> 87

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 87

Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 88

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 88

Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

1 5 10

<210> 89

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 89

Ser Tyr Asp Gly Ser Asn

1 5

<210> 90

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 90

Gly Phe Thr Phe Ser Ser Tyr Gly

1 5

<210> 91

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 91

Ile Ser Tyr Asp Gly Ser Asn Lys

1 5

<210> 92

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 92

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

1 5 10 15

<210> 93

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 93

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 94

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 94

caagtgcagc tgcaggaatc cggtggcgga gtcgtgcagc ctggaaggag cctgagactc 60

tcatgcgccg cgtcagggtt caccttttcc tcctacggga tgcattgggt cagacaggcc 120

cccggaaagg gactcgaatg ggtggctgtg atcagctacg acggctccaa caagtactac 180

gccgactccg tgaaaggccg gttcactatc tcccgggaca actccaagaa cacgctgtat 240

ctgcaaatga attcactgcg cgcggaggat accgctgtgt actactgcgg tggctccggt 300

tacgccctgc acgatgacta ttacggcctt gacgtctggg gccagggaac cctcgtgact 360

gtgtccagc 369

<210> 95

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 95

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 96

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 96

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 97

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 97

Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr Val

1 5 10

<210> 98

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 98

Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr

1 5 10

<210> 99

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 99

Asp Val Ser

1

<210> 100

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 100

Tyr Thr Ser Ser Ser Thr Leu Tyr

1 5

<210> 101

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 101

Ser Ser Asp Val Gly Gly Tyr Asn Tyr

1 5

<210> 102

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 102

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 103

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 103

cagagcgcac tgactcagcc ggcatccgtg tccggtagcc ccggacagtc gattaccatc 60

tcctgtaccg gcacctcctc cgacgtggga gggtacaact acgtgtcgtg gtaccagcag 120

cacccaggaa aggcccctaa gttgatgatc tacgatgtgt caaaccgccc gtctggagtc 180

tccaaccggt tctccggctc caagtccggc aacaccgcca gcctgaccat tagcgggctg 240

caagccgagg atgaggccga ctactactgc tcgagctaca catcctcgag caccctctac 300

gtgttcggct cggggactaa ggtcaccgtg ctg 333

<210> 104

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 104

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 105

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 105

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln

130 135 140

Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys

145 150 155 160

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr

165 170 175

Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser

180 185 190

Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly

195 200 205

Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala

210 215 220

Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr Val Phe

225 230 235 240

Gly Ser Gly Thr Lys Val Thr Val Leu

245

<210> 106

<211> 747

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 106

caagtgcagc tgcaggaatc cggtggcgga gtcgtgcagc ctggaaggag cctgagactc 60

tcatgcgccg cgtcagggtt caccttttcc tcctacggga tgcattgggt cagacaggcc 120

cccggaaagg gactcgaatg ggtggctgtg atcagctacg acggctccaa caagtactac 180

gccgactccg tgaaaggccg gttcactatc tcccgggaca actccaagaa cacgctgtat 240

ctgcaaatga attcactgcg cgcggaggat accgctgtgt actactgcgg tggctccggt 300

tacgccctgc acgatgacta ttacggcctt gacgtctggg gccagggaac cctcgtgact 360

gtgtccagcg gtggaggagg ttcgggcgga ggagatcag gagggggtgg atcgcagagc 420

gcactgactc agccggcatc cgtgtccggt agccccggac agtcgattac catctcctgt 480

accggcacct cctccgacgt gggagggtac aactacgtgt cgtggtacca gcagcaccca 540

ggaaaggccc ctaagttgat gatctacgat gtgtcaaacc gcccgtctgg agtctccaac 600

cggttctccg gctccaagtc cggcaacacc gccagcctga ccattagcgg gctgcaagcc 660

gaggatgagg ccgactacta ctgctcgagc tacacatcct cgagcaccct ctacgtgttc 720

ggctcgggga ctaaggtcac cgtgctg 747

<210> 107

<211> 472

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 107

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln

130 135 140

Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys

145 150 155 160

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr

165 170 175

Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser

180 185 190

Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly

195 200 205

Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala

210 215 220

Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr Val Phe

225 230 235 240

Gly Ser Gly Thr Lys Val Thr Val Leu Thr Thr Thr Pro Ala Pro Arg

245 250 255

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

260 265 270

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

275 280 285

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

290 295 300

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

305 310 315 320

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

325 330 335

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

340 345 350

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

355 360 365

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

370 375 380

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

385 390 395 400

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

405 410 415

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

420 425 430

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

435 440 445

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

450 455 460

His Met Gln Ala Leu Pro Pro Arg

465 470

<210> 108

<211> 1416

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 108

caagtgcagc tgcaggaatc cggtggcgga gtcgtgcagc ctggaaggag cctgagactc 60

tcatgcgccg cgtcagggtt caccttttcc tcctacggga tgcattgggt cagacaggcc 120

cccggaaagg gactcgaatg ggtggctgtg atcagctacg acggctccaa caagtactac 180

gccgactccg tgaaaggccg gttcactatc tcccgggaca actccaagaa cacgctgtat 240

ctgcaaatga attcactgcg cgcggaggat accgctgtgt actactgcgg tggctccggt 300

tacgccctgc acgatgacta ttacggcctt gacgtctggg gccagggaac cctcgtgact 360

gtgtccagcg gtggaggagg ttcgggcgga ggagatcag gagggggtgg atcgcagagc 420

gcactgactc agccggcatc cgtgtccggt agccccggac agtcgattac catctcctgt 480

accggcacct cctccgacgt gggagggtac aactacgtgt cgtggtacca gcagcaccca 540

ggaaaggccc ctaagttgat gatctacgat gtgtcaaacc gcccgtctgg agtctccaac 600

cggttctccg gctccaagtc cggcaacacc gccagcctga ccattagcgg gctgcaagcc 660

gaggatgagg ccgactacta ctgctcgagc tacacatcct cgagcaccct ctacgtgttc 720

ggctcgggga ctaaggtcac cgtgctgacc actaccccag caccgaggcc acccaccccg 780

gctcctacca tcgcctccca gcctctgtcc ctgcgtccgg aggcatgtag acccgcagct 840

ggtggggccg tgcatacccg gggtcttgac ttcgcctgcg atatctacat ttgggcccct 900

ctggctggta cttgcggggt cctgctgctt tcactcgtga tcactcttta ctgtaagcgc 960

ggtcggaaga agctgctgta catctttaag caacccttca tgaggcctgt gcagactact 1020

caagaggagg acggctgttc atgccggttc ccagaggagg aggaaggcgg ctgcgaactg 1080

cgcgtgaaat tcagccgcag cgcagatgct ccagcctacc agcaggggca gaaccagctc 1140

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 1200

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 1260

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 1320

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 1380

tatgacgctc ttcacatgca ggccctgccg cctcgg 1416

<210> 109

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 109

Val Ile Ser Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 110

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 110

Ser Tyr Lys Gly Ser Asn

1 5

<210> 111

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 111

Ile Ser Tyr Lys Gly Ser Asn Lys

1 5

<210> 112

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 112

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 113

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 113

caagtgcagc ttgtcgaatc gggaggcgga gtggtgcagc ctggacgatc gctccggctc 60

tcatgtgccg cgagcggatt caccttctcg agctacggca tgcactgggt cagacaagcc 120

caggaaagg gcctggaatg ggtggctgtc atctcgtaca agggctcaaa caagtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgcgata actccaagaa taccctctat 240

ctgcaaatga acagcctgag ggccgaggat actgcagtgt actactgcgg gggttcaggc 300

tacgcgctgc acgacgacta ctacggattg gacgtctggg gccaaggaac tcttgtgacc 360

gtgtcctct 369

<210> 114

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 114

Glu Val Ser Asn Arg Leu Arg

1 5

<210> 115

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 115

Ser Ser Tyr Thr Ser Ser Ser Ala Leu Tyr Val

1 5 10

<210> 116

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 116

Glu Val Ser

1

<210> 117

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 117

Tyr Thr Ser Ser Ser Ala Leu Tyr

1 5

<210> 118

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 118

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Leu Arg Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Ala Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 119

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 119

cagagcgcgc tgactcagcc tgcctccgtg agcggttcgc cgggacagtc cattaccatt 60

tcgtgcaccg ggacctcctc cgacgtggga ggctacaact acgtgtcctg gtaccagcag 120

catcccggaa aggccccgaa gctgatgatc tacgaagtgt cgaacagact gcggggagtc 180

tccaaccgct tttccggggtc caagtccggc aacaccgcca gcctgaccat cagcgggctc 240

caggcagaag atgaggctga ctattactgc tcctcctaca cgtcaagctc cgccctctac 300

gtgttcgggt ccgggaccaa agtcactgtg ctg 333

<210> 120

<211> 254

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 120

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

145 150 155 160

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

165 170 175

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

180 185 190

Ile Tyr Glu Val Ser Asn Arg Leu Arg Gly Val Ser Asn Arg Phe Ser

195 200 205

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

210 215 220

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

225 230 235 240

Ala Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu

245 250

<210> 121

<211> 762

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 121

caagtgcagc ttgtcgaatc gggaggcgga gtggtgcagc ctggacgatc gctccggctc 60

tcatgtgccg cgagcggatt caccttctcg agctacggca tgcactgggt cagacaagcc 120

caggaaagg gcctggaatg ggtggctgtc atctcgtaca agggctcaaa caagtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgcgata actccaagaa taccctctat 240

ctgcaaatga acagcctgag ggccgaggat actgcagtgt actactgcgg gggttcaggc 300

tacgcgctgc acgacgacta ctacggattg gacgtctggg gccaaggaac tcttgtgacc 360

gtgtcctctg gtggaggcgg atcaggggggt ggcggatctg ggggtggtgg ttccggggga 420

ggaggatcgc agagcgcgct gactcagcct gcctccgtga gcggttcgcc gggacagtcc 480

attaccattt cgtgcaccgg gacctcctcc gacgtgggag gctacaacta cgtgtcctgg 540

taccagcagc atcccggaaa ggccccgaag ctgatgatct acgaagtgtc gaacagactg 600

cggggagtct ccaaccgctt ttccgggtcc aagtccggca acaccgccag cctgaccatc 660

agcgggctcc aggcagaaga tgaggctgac tattactgct cctcctacac gtcaagctcc 720

gccctctacg tgttcgggtc cgggaccaaa gtcactgtgc tg 762

<210> 122

<211> 477

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 122

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

145 150 155 160

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

165 170 175

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

180 185 190

Ile Tyr Glu Val Ser Asn Arg Leu Arg Gly Val Ser Asn Arg Phe Ser

195 200 205

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

210 215 220

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

225 230 235 240

Ala Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Thr Thr

245 250 255

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

260 265 270

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

275 280 285

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

290 295 300

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

305 310 315 320

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

325 330 335

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

340 345 350

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

355 360 365

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

370 375 380

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

385 390 395 400

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

405 410 415

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

420 425 430

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

435 440 445

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

450 455 460

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475

<210> 123

<211> 1431

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 123

caagtgcagc ttgtcgaatc gggaggcgga gtggtgcagc ctggacgatc gctccggctc 60

tcatgtgccg cgagcggatt caccttctcg agctacggca tgcactgggt cagacaagcc 120

caggaaagg gcctggaatg ggtggctgtc atctcgtaca agggctcaaa caagtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgcgata actccaagaa taccctctat 240

ctgcaaatga acagcctgag ggccgaggat actgcagtgt actactgcgg gggttcaggc 300

tacgcgctgc acgacgacta ctacggattg gacgtctggg gccaaggaac tcttgtgacc 360

gtgtcctctg gtggaggcgg atcaggggggt ggcggatctg ggggtggtgg ttccggggga 420

ggaggatcgc agagcgcgct gactcagcct gcctccgtga gcggttcgcc gggacagtcc 480

attaccattt cgtgcaccgg gacctcctcc gacgtgggag gctacaacta cgtgtcctgg 540

taccagcagc atcccggaaa ggccccgaag ctgatgatct acgaagtgtc gaacagactg 600

cggggagtct ccaaccgctt ttccggggtcc aagtccggca acaccgccag cctgaccatc 660

agcgggctcc aggcagaaga tgaggctgac tattactgct cctcctacac gtcaagctcc 720

gccctctacg tgttcgggtc cgggaccaaa gtcactgtgc tgaccactac cccagcaccg 780

aggccaccca ccccggctcc taccatcgcc tcccagcctc tgtccctgcg tccggaggca 840

tgtagacccg cagctggtgg ggccgtgcat acccggggtc ttgacttcgc ctgcgatatc 900

tacatttggg cccctctggc tggtacttgc ggggtcctgc tgctttcact cgtgatcact 960

ctttactgta agcgcggtcg gaagaagctg ctgtacatct ttaagcaacc cttcatgagg 1020

cctgtgcaga ctactcaaga ggagaggacggc tgttcatgcc ggttcccaga ggagggaggaa 1080

ggcggctgcg aactgcgcgt gaaattcagc cgcagcgcag atgctccagc ctaccagcag 1140

gggcagaacc agctctacaa cgaactcaat cttggtcgga gagaggagta cgacgtgctg 1200

gacaagcgga gaggacggga cccagaaatg ggcgggaagc cgcgcagaaa gaatccccaa 1260

gagggcctgt acaacgagct ccaaaaggat aagatggcag aagcctatag cgagattggt 1320

atgaaagggg aacgcagaag aggcaaaggc cacgacggac tgtaccaggg actcagcacc 1380

gccaccaagg acacctatga cgctcttcac atgcaggccc tgccgcctcg g 1431

<210> 124

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 124

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Leu Arg Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 125

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 125

cagagcgcgc tgactcagcc tgcctccgtg agcggttcgc cgggacagtc cattaccatt 60

tcgtgcaccg ggacctcctc cgacgtggga ggctacaact acgtgtcctg gtaccagcag 120

catcccggaa aggccccgaa gctgatgatc tacgaagtgt cgaacagact gcggggagtc 180

tccaaccgct tttccggggtc caagtccggc aacaccgcca gcctgaccat cagcgggctc 240

caggcagaag atgaggctga ctattactgc tcctcctaca cgtcaagctc caccctctac 300

gtgttcgggt ccgggaccaa agtcactgtg ctg 333

<210> 126

<211> 254

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 126

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

145 150 155 160

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

165 170 175

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

180 185 190

Ile Tyr Glu Val Ser Asn Arg Leu Arg Gly Val Ser Asn Arg Phe Ser

195 200 205

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

210 215 220

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

225 230 235 240

Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu

245 250

<210> 127

<211> 762

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 127

caagtgcagc ttgtcgaatc gggaggcgga gtggtgcagc ctggacgatc gctccggctc 60

tcatgtgccg cgagcggatt caccttctcg agctacggca tgcactgggt cagacaagcc 120

caggaaagg gcctggaatg ggtggctgtc atctcgtaca agggctcaaa caagtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgcgata actccaagaa taccctctat 240

ctgcaaatga acagcctgag ggccgaggat actgcagtgt actactgcgg gggttcaggc 300

tacgcgctgc acgacgacta ctacggattg gacgtctggg gccaaggaac tcttgtgacc 360

gtgtcctctg gtggaggcgg atcaggggggt ggcggatctg ggggtggtgg ttccggggga 420

ggaggatcgc agagcgcgct gactcagcct gcctccgtga gcggttcgcc gggacagtcc 480

attaccattt cgtgcaccgg gacctcctcc gacgtgggag gctacaacta cgtgtcctgg 540

taccagcagc atcccggaaa ggccccgaag ctgatgatct acgaagtgtc gaacagactg 600

cggggagtct ccaaccgctt ttccggggtcc aagtccggca acaccgccag cctgaccatc 660

agcgggctcc aggcagaaga tgaggctgac tattactgct cctcctacac gtcaagctcc 720

accctctacg tgttcgggtc cgggaccaaa gtcactgtgc tg 762

<210> 128

<211> 477

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 128

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

145 150 155 160

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

165 170 175

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

180 185 190

Ile Tyr Glu Val Ser Asn Arg Leu Arg Gly Val Ser Asn Arg Phe Ser

195 200 205

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

210 215 220

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

225 230 235 240

Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Thr Thr

245 250 255

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

260 265 270

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

275 280 285

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

290 295 300

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

305 310 315 320

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

325 330 335

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

340 345 350

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

355 360 365

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

370 375 380

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

385 390 395 400

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

405 410 415

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

420 425 430

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

435 440 445

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

450 455 460

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475

<210> 129

<211> 1431

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 129

caagtgcagc ttgtcgaatc gggaggcgga gtggtgcagc ctggacgatc gctccggctc 60

tcatgtgccg cgagcggatt caccttctcg agctacggca tgcactgggt cagacaagcc 120

caggaaagg gcctggaatg ggtggctgtc atctcgtaca agggctcaaa caagtactac 180

gccgactccg tgaagggccg gttcaccatc tcccgcgata actccaagaa taccctctat 240

ctgcaaatga acagcctgag ggccgaggat actgcagtgt actactgcgg gggttcaggc 300

tacgcgctgc acgacgacta ctacggattg gacgtctggg gccaaggaac tcttgtgacc 360

gtgtcctctg gtggaggcgg atcaggggggt ggcggatctg ggggtggtgg ttccggggga 420

ggaggatcgc agagcgcgct gactcagcct gcctccgtga gcggttcgcc gggacagtcc 480

attaccattt cgtgcaccgg gacctcctcc gacgtgggag gctacaacta cgtgtcctgg 540

taccagcagc atcccggaaa ggccccgaag ctgatgatct acgaagtgtc gaacagactg 600

cggggagtct ccaaccgctt ttccgggtcc aagtccggca acaccgccag cctgaccatc 660

agcgggctcc aggcagaaga tgaggctgac tattactgct cctcctacac gtcaagctcc 720

accctctacg tgttcgggtc cgggaccaaa gtcactgtgc tgaccactac cccagcaccg 780

aggccaccca ccccggctcc taccatcgcc tcccagcctc tgtccctgcg tccggaggca 840

tgtagacccg cagctggtgg ggccgtgcat acccggggtc ttgacttcgc ctgcgatatc 900

tacatttggg cccctctggc tggtacttgc ggggtcctgc tgctttcact cgtgatcact 960

ctttactgta agcgcggtcg gaagaagctg ctgtacatct ttaagcaacc cttcatgagg 1020

cctgtgcaga ctactcaaga ggagaggacggc tgttcatgcc ggttcccaga ggagggaggaa 1080

ggcggctgcg aactgcgcgt gaaattcagc cgcagcgcag atgctccagc ctaccagcag 1140

gggcagaacc agctctacaa cgaactcaat cttggtcgga gagaggagta cgacgtgctg 1200

gacaagcgga gaggacggga cccagaaatg ggcgggaagc cgcgcagaaa gaatccccaa 1260

gagggcctgt acaacgagct ccaaaaggat aagatggcag aagcctatag cgagattggt 1320

atgaaagggg aacgcagaag aggcaaaggc cacgacggac tgtaccaggg actcagcacc 1380

gccaccaagg acacctatga cgctcttcac atgcaggccc tgccgcctcg g 1431

<210> 130

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace="K"

<220>

<221> SITE

<222> (1)..(17)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 130

Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 131

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="E"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="L"

<220>

<221> VARIANT

<222> (7)..(7)

<223> /replace="R"

<220>

<221> SITE

<222> (1)..(7)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 131

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 132

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace="A"

<220>

<221> SITE

<222> (1)..(11)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 132

Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr Val

1 5 10

<210> 133

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (3)..(3)

<223> /replace="K"

<220>

<221> SITE

<222> (1)..(6)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 133

Ser Tyr Asp Gly Ser Asn

1 5

<210> 134

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="E"

<220>

<221> SITE

<222> (1)..(3)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 134

Asp Val Ser

1

<210> 135

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="A"

<220>

<221> SITE

<222> (1)..(8)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 135

Tyr Thr Ser Ser Ser Thr Leu Tyr

1 5

<210> 136

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace="K"

<220>

<221> SITE

<222> (1)..(8)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 136

Ile Ser Tyr Asp Gly Ser Asn Lys

1 5

<210> 137

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 137

Gly Phe Trp Met Ser

1 5

<210> 138

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 138

Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Arg

1 5 10 15

Gly

<210> 139

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 139

Ala Leu Asp Tyr Tyr Gly Met Asp Val

1 5

<210> 140

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 140

Gly Phe Thr Phe Ser Gly Phe

1 5

<210> 141

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 141

Lys Gln Asp Gly Ser Glu

1 5

<210> 142

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 142

Gly Phe Thr Phe Ser Gly Phe Trp

1 5

<210> 143

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 143

Ile Lys Gln Asp Gly Ser Glu Lys

1 5

<210> 144

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 144

Ala Arg Ala Leu Asp Tyr Tyr Gly Met Asp Val

1 5 10

<210> 145

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 145

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Phe

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Leu Asp Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 146

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 146

gaagtgcaac tggtggagag cggtggaggg cttgtccagc ccggaggatc gctgcggctg 60

tcctgtgctg cgtccgggtt caccttctcc ggcttctgga tgtcctgggt cagacaggca 120

ccgggaaagg gcctcgaatg ggtggccaac atcaagcagg atggctccga gaagtactac 180

gtcgactccg tgagaggccg cttcaccatc tcccgggaca acgccaagaa ctcgctgtac 240

ctccaaatga atagcctcag ggcggaagat actgctgtgt attactgcgc acgcgccctt 300

gactactacg gcatggacgt ctggggccaa gggaccactg tgaccgtgtc tagc 354

<210> 147

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 147

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu

1 5 10 15

Asp

<210> 148

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 148

Thr Leu Ser Tyr Arg Ala Ser

1 5

<210> 149

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 149

Thr Gln Arg Leu Glu Phe Pro Ser Ile Thr

1 5 10

<210> 150

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 150

Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

1 5 10

<210> 151

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 151

Thr Leu Ser

1

<210> 152

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 152

Arg Leu Glu Phe Pro Ser Ile

1 5

<210> 153

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 153

Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

1 5 10

<210> 154

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 154

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Arg Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Thr Gln

85 90 95

Arg Leu Glu Phe Pro Ser Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu

100 105 110

Ile Lys

<210> 155

<211> 342

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 155

gatatcgtga tgacccagac tcccctgtcc ctgcctgtga ctcccggaga accagcctcc 60

atttcctgcc ggtcctccca gtccctgctg gacagcgacg acggcaacac ttacctggac 120

tggtacttgc agaagccggg ccaatcgcct cgcctgctga tctataccct gtcataccgg 180

gcctcaggag tgcctgaccg cttctcggga tcagggagcg ggaccgattt caccctgaaa 240

atttcccgag tggaagccga ggacgtcgga ctgtactact gcacccagcg cctcgaattc 300

ccgtcgatta cgtttggaca gggtacccgg cttgagatca ag 342

<210> 156

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 156

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Phe

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Leu Asp Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

130 135 140

Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

165 170 175

Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

180 185 190

Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

210 215 220

Glu Asp Val Gly Leu Tyr Tyr Cys Thr Gln Arg Leu Glu Phe Pro Ser

225 230 235 240

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

245 250

<210> 157

<211> 756

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 157

gaagtgcaac tggtggagag cggtggaggg cttgtccagc ccggaggatc gctgcggctg 60

tcctgtgctg cgtccgggtt caccttctcc ggcttctgga tgtcctgggt cagacaggca 120

ccgggaaagg gcctcgaatg ggtggccaac atcaagcagg atggctccga gaagtactac 180

gtcgactccg tgagaggccg cttcaccatc tcccgggaca acgccaagaa ctcgctgtac 240

ctccaaatga atagcctcag ggcggaagat actgctgtgt attactgcgc acgcgccctt 300

gactactacg gcatggacgt ctggggccaa gggaccactg tgaccgtgtc tagcggaggc 360

ggaggttcag ggggcggtgg atcaggcgga ggaggatcgg ggggtggtgg atcggatatc 420

gtgatgaccc agactcccct gtccctgcct gtgactcccg gagaaccagc ctccatttcc 480

tgccggtcct cccagtccct gctggacagc gacgacggca acacttacct ggactggtac 540

ttgcagaagc cgggccaatc gcctcgcctg ctgatctata ccctgtcata ccgggcctca 600

ggagtgcctg accgcttctc gggatcaggg agcgggaccg atttcaccct gaaaatttcc 660

cgagtggaag ccgaggacgt cggactgtac tactgcaccc agcgcctcga attcccgtcg 720

attacgtttg gacagggtac ccggcttgag atcaag 756

<210> 158

<211> 475

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 158

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Phe

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Leu Asp Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

130 135 140

Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

165 170 175

Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

180 185 190

Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

210 215 220

Glu Asp Val Gly Leu Tyr Tyr Cys Thr Gln Arg Leu Glu Phe Pro Ser

225 230 235 240

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Thr Thr Thr Pro

245 250 255

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

260 265 270

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

275 280 285

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

290 295 300

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

305 310 315 320

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

325 330 335

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

340 345 350

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

355 360 365

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

370 375 380

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

385 390 395 400

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

405 410 415

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

420 425 430

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

435 440 445

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

450 455 460

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475

<210> 159

<211> 1425

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 159

gaagtgcaac tggtggagag cggtggaggg cttgtccagc ccggaggatc gctgcggctg 60

tcctgtgctg cgtccgggtt caccttctcc ggcttctgga tgtcctgggt cagacaggca 120

ccgggaaagg gcctcgaatg ggtggccaac atcaagcagg atggctccga gaagtactac 180

gtcgactccg tgagaggccg cttcaccatc tcccgggaca acgccaagaa ctcgctgtac 240

ctccaaatga atagcctcag ggcggaagat actgctgtgt attactgcgc acgcgccctt 300

gactactacg gcatggacgt ctggggccaa gggaccactg tgaccgtgtc tagcggaggc 360

ggaggttcag ggggcggtgg atcaggcgga ggaggatcgg ggggtggtgg atcggatatc 420

gtgatgaccc agactcccct gtccctgcct gtgactcccg gagaaccagc ctccatttcc 480

tgccggtcct cccagtccct gctggacagc gacgacggca acacttacct ggactggtac 540

ttgcagaagc cgggccaatc gcctcgcctg ctgatctata ccctgtcata ccgggcctca 600

ggagtgcctg accgcttctc gggatcaggg agcgggaccg atttcaccct gaaaatttcc 660

cgagtggaag ccgaggacgt cggactgtac tactgcaccc agcgcctcga attcccgtcg 720

attacgtttg gacagggtac ccggcttgag atcaagacca ctaccccagc accgaggcca 780

cccaccccgg ctcctaccat cgcctcccag cctctgtccc tgcgtccgga ggcatgtaga 840

cccgcagctg gtggggccgt gcatacccgg ggtcttgact tcgcctgcga tatctacatt 900

tgggcccctc tggctggtac ttgcggggtc ctgctgcttt cactcgtgat cactctttac 960

tgtaagcgcg gtcggaagaa gctgctgtac atctttaagc aacccttcat gaggcctgtg 1020

cagactactc aagaggagga cggctgttca tgccggttcc cagaggagga ggaaggcggc 1080

tgcgaactgc gcgtgaaatt cagccgcagc gcagatgctc cagcctacca gcaggggcag 1140

aaccagctct acaacgaact caatcttggt cggagagagg agtacgacgt gctggacaag 1200

cggagaggac gggacccaga aatgggcggg aagccgcgca gaaagaatcc ccaagaggc 1260

ctgtacaacg agctccaaaa ggataagatg gcagaagcct atagcgagat tggtatgaaa 1320

ggggaacgca gaagaggcaa aggccacgac ggactgtacc agggactcag caccgccacc 1380

aaggacacct atgacgctct tcacatgcag gccctgccgc ctcgg 1425

<210> 160

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 160

Ser Phe Arg Met Asn

1 5

<210> 161

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 161

Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 162

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 162

Trp Leu Ser Tyr Tyr Gly Met Asp Val

1 5

<210> 163

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 163

Gly Phe Thr Phe Ser Ser Phe

1 5

<210> 164

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 164

Ser Ser Ser Ser Ser Tyr

1 5

<210> 165

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 165

Gly Phe Thr Phe Ser Ser Phe Arg

1 5

<210> 166

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 166

Ile Ser Ser Ser Ser Ser Tyr Ile

1 5

<210> 167

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 167

Ala Arg Trp Leu Ser Tyr Tyr Gly Met Asp Val

1 5 10

<210> 168

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 168

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Arg Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Leu Ser Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 169

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 169

gaagtgcaac tggtggagag cggtggaggg cttgtcaagc ccggaggatc gctgcggctg 60

tcctgtgctg cgtccgggtt caccttctcc tcgttccgca tgaactgggt cagacaggca 120

ccgggaaagg gcctcgaatg ggtgtcctca atctcatcgt cctcgtccta catctactac 180

gccgactccg tgaaaggccg cttcaccatc tcccgggaca acgccaagaa ctcgctgtac 240

ctccaaatga atagcctcag ggcggaagat actgctgtgt attactgcgc acgctggctt 300

tcctactacg gcatggacgt ctggggccaa gggaccactg tgaccgtgtc tagc 354

<210> 170

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 170

Thr Leu Ser Phe Arg Ala Ser

1 5

<210> 171

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 171

Met Gln Arg Ile Gly Phe Pro Ile Thr

1 5

<210> 172

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 172

Arg Ile Gly Phe Pro Ile

1 5

<210> 173

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 173

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Phe Arg Ala Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Arg Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln

85 90 95

Arg Ile Gly Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile

100 105 110

Lys

<210> 174

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 174

gatatcgtga tgacccagac tcccctgtcc ctgcctgtga ctcccggaga accagcctcc 60

atttcctgcc ggtcctccca gtccctgctg gacagcgacg acggcaacac ttacctggac 120

tggtacttgc agaagccggg ccaatcgcct cagctgctga tctataccct gtcattccgg 180

gcctcaggag tgcctgaccg cttctcggga tcagggagcg ggaccgattt caccctgaaa 240

attaggcgag tggaagccga ggacgtcgga gtgtactact gcatgcagcg catcggcttc 300

ccgattacgt ttggacaggg tacccggctt gagatcaag 339

<210> 175

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 175

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Arg Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Leu Ser Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

130 135 140

Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

165 170 175

Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

180 185 190

Tyr Thr Leu Ser Phe Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Arg Arg Val Glu Ala

210 215 220

Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Arg Ile Gly Phe Pro Ile

225 230 235 240

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

245 250

<210> 176

<211> 753

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 176

gaagtgcaac tggtggagag cggtggaggg cttgtcaagc ccggaggatc gctgcggctg 60

tcctgtgctg cgtccgggtt caccttctcc tcgttccgca tgaactgggt cagacaggca 120

ccgggaaagg gcctcgaatg ggtgtcctca atctcatcgt cctcgtccta catctactac 180

gccgactccg tgaaaggccg cttcaccatc tcccgggaca acgccaagaa ctcgctgtac 240

ctccaaatga atagcctcag ggcggaagat actgctgtgt attactgcgc acgctggctt 300

tcctactacg gcatggacgt ctggggccaa gggaccactg tgaccgtgtc tagcggaggc 360

ggaggttcag ggggcggtgg atcaggcgga ggaggatcgg ggggtggtgg atcggatatc 420

gtgatgaccc agactcccct gtccctgcct gtgactcccg gagaaccagc ctccatttcc 480

tgccggtcct cccagtccct gctggacagc gacgacggca acacttacct ggactggtac 540

ttgcagaagc cgggccaatc gcctcagctg ctgatctata ccctgtcatt ccgggcctca 600

ggagtgcctg accgcttctc gggatcaggg agcgggaccg atttcaccct gaaaattagg 660

cgagtggaag ccgaggacgt cggagtgtac tactgcatgc agcgcatcgg cttcccgatt 720

acgtttggac agggtacccg gcttgagatc aag 753

<210> 177

<211> 474

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 177

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Arg Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Leu Ser Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

130 135 140

Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

145 150 155 160

Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr

165 170 175

Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

180 185 190

Tyr Thr Leu Ser Phe Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Arg Arg Val Glu Ala

210 215 220

Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Arg Ile Gly Phe Pro Ile

225 230 235 240

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Thr Thr Thr Pro Ala

245 250 255

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

260 265 270

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

275 280 285

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

290 295 300

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

305 310 315 320

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

325 330 335

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

340 345 350

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

355 360 365

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

370 375 380

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

385 390 395 400

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

405 410 415

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

420 425 430

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

435 440 445

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

450 455 460

Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470

<210> 178

<211> 1422

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 178

gaagtgcaac tggtggagag cggtggaggg cttgtcaagc ccggaggatc gctgcggctg 60

tcctgtgctg cgtccgggtt caccttctcc tcgttccgca tgaactgggt cagacaggca 120

ccgggaaagg gcctcgaatg ggtgtcctca atctcatcgt cctcgtccta catctactac 180

gccgactccg tgaaaggccg cttcaccatc tcccgggaca acgccaagaa ctcgctgtac 240

ctccaaatga atagcctcag ggcggaagat actgctgtgt attactgcgc acgctggctt 300

tcctactacg gcatggacgt ctggggccaa gggaccactg tgaccgtgtc tagcggaggc 360

ggaggttcag ggggcggtgg atcaggcgga ggaggatcgg ggggtggtgg atcggatatc 420

gtgatgaccc agactcccct gtccctgcct gtgactcccg gagaaccagc ctccatttcc 480

tgccggtcct cccagtccct gctggacagc gacgacggca acacttacct ggactggtac 540

ttgcagaagc cgggccaatc gcctcagctg ctgatctata ccctgtcatt ccgggcctca 600

ggagtgcctg accgcttctc gggatcaggg agcgggaccg atttcaccct gaaaattagg 660

cgagtggaag ccgaggacgt cggagtgtac tactgcatgc agcgcatcgg cttcccgatt 720

acgtttggac agggtacccg gcttgagatc aagaccacta ccccagcacc gaggccaccc 780

accccggctc ctaccatcgc ctcccagcct ctgtccctgc gtccggaggc atgtagaccc 840

gcagctggtg gggccgtgca tacccggggt cttgacttcg cctgcgatat ctacatttgg 900

gcccctctgg ctggtacttg cggggtcctg ctgctttcac tcgtgatcac tctttactgt 960

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 1020

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 1080

gaactgcgcg tgaaattcag ccgcagcgca gatgctccag cctaccagca ggggcagaac 1140

cagctctaca acgaactcaa tcttggtcgg agagaggagt acgacgtgct ggacaagcgg 1200

agaggacggg acccagaaat gggcgggaag ccgcgcagaa agaatcccca agaggcctg 1260

tacaacgagc tccaaaagga taagatggca gaagcctata gcgagattgg tatgaaaggg 1320

gaacgcagaa gaggcaaagg ccacgacgga ctgtaccagg gactcagcac cgccaccaag 1380

gacacctatg acgctcttca catgcaggcc ctgccgcctc gg 1422

<210> 179

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="S"

<220>

<221> VARIANT

<222> (3)..(3)

<223> /replace="R"

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace="N"

<220>

<221> SITE

<222> (1)..(5)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 179

Gly Phe Trp Met Ser

1 5

<210> 180

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="S"

<220>

<221> VARIANT

<222> (3)..(3)

<223> /replace="S"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace="S"

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace="S"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="S"

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace="Y"

<220>

<221> VARIANT

<222> (9)..(9)

<223> /replace="I"

<220>

<221> VARIANT

<222> (12)..(12)

<223> /replace="A"

<220>

<221> VARIANT

<222> (16)..(16)

<223> /replace="K"

<220>

<221> SITE

<222> (1)..(17)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 180

Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Arg

1 5 10 15

Gly

<210> 181

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="W"

<220>

<221> VARIANT

<222> (3)..(3)

<223> /replace="S"

<220>

<221> SITE

<222> (1)..(9)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 181

Ala Leu Asp Tyr Tyr Gly Met Asp Val

1 5

<210> 182

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace="F"

<220>

<221> SITE

<222> (1)..(7)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 182

Thr Leu Ser Tyr Arg Ala Ser

1 5

<210> 183

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="M"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace="I"

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace="G"

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace=" "

<220>

<221> SITE

<222> (1)..(10)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 183

Thr Gln Arg Leu Glu Phe Pro Ser Ile Thr

1 5 10

<210> 184

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="S"

<220>

<221> SITE

<222> (1)..(7)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 184

Gly Phe Thr Phe Ser Gly Phe

1 5

<210> 185

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (1)..(1)

<223> /replace="S"

<220>

<221> VARIANT

<222> (2)..(2)

<223> /replace="S"

<220>

<221>MOD_RES

<222> (3)..(3)

<223> /replace="S"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace="S"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="Y"

<220>

<221> SITE

<222> (1)..(6)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 185

Lys Gln Asp Gly Ser Glu

1 5

<210> 186

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (2)..(2)

<223> /replace="I"

<220>

<221> VARIANT

<222> (3)..(3)

<223> /replace="G"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace=" "

<220>

<221> SITE

<222> (1)..(7)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 186

Arg Leu Glu Phe Pro Ser Ile

1 5

<210> 187

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (6)..(6)

<223> /replace="S"

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace="R"

<220>

<221> SITE

<222> (1)..(8)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 187

Gly Phe Thr Phe Ser Gly Phe Trp

1 5

<210> 188

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (2)..(2)

<223> /replace="K"

<220>

<221>MOD_RES

<222> (3)..(3)

<223> /replace="S"

<220>

<221> VARIANT

<222> (4)..(4)

<223> /replace="S"

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace="S"

<220>

<221> VARIANT

<222> (7)..(7)

<223> /replace="Y"

<220>

<221> VARIANT

<222> (8)..(8)

<223> /replace="I"

<220>

<221> SITE

<222> (1)..(8)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 188

Ile Lys Gln Asp Gly Ser Glu Lys

1 5

<210> 189

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> VARIANT

<222> (3)..(3)

<223> /replace="W"

<220>

<221> VARIANT

<222> (5)..(5)

<223> /replace="S"

<220>

<221> SITE

<222> (1)..(11)

<223> /note="Variant residues given in the sequence have no

preference with respect to those in the annotations

for variant positions"

<400> 189

Ala Arg Ala Leu Asp Tyr Tyr Gly Met Asp Val

1 5 10

<210> 190

<211> 521

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

PGK Promoter sequence"

<400> 190

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc ggccccccgg gtgttcccat 360

cgccgcttct aggcccactg cgacgcttgc ctgcacttct tacacgctct gggtcccagc 420

cgcggcgacg caaagggcct tggtgcgggt ctcgtcggcg cagggacgcg tttgggtccc 480

gacggaacct tttccgcgtt ggggttgggg caccataagc t 521

<210> 191

<211> 221

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 191

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac g 221

<210> 192

<211> 324

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 192

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccg 324

<210> 193

<211> 422

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 193

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtgtg 120

gggcggaggg cgtggcgggg aagggccggc gacgagagcc gcgcgggacg actcgtcggc 180

gataaccggt gtcgggtagc gccagccgcg cgacggtaac gagggaccgc gacaggcaga 240

cgctcccatg atcactctgc acgccgaagg caaatagtgc aggccgtgcg gcgcttggcg 300

ttccttggaa gggctgaatc cccgcctcgt ccttcgcagc ggccccccgg gtgttcccat 360

cgccgcttct aggcccactg cgacgcttgc ctgcacttct tacacgctct gggtcccagc 420

cg 422

<210> 194

<400> 194

000

<210> 195

<400> 195

000

<210> 196

<400> 196

000

<210> 197

<400> 197

000

<210> 198

<211> 118

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 198

acccctctct ccagccacta agccagttgc tccctcggct gacggctgca cgcgaggcct 60

ccgaacgtct tacgccttgt ggcgcgcccg tccttgtccc gggtgtgatg gcggggtg 118

<210> 199

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 199

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 200

<211> 239

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 200

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ala Leu Ser Asn His

20 25 30

Gly Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr Ala Ala Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser

85 90 95

Ala His Gly Gly Glu Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr

210 215 220

Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

225 230 235

<210> 201

<211> 717

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 201

gaagtgcaat tggtggaatc agggggagga cttgtgcagc ctggaggatc gctgagactg 60

tcatgtgccg tgtccggctt tgccctgtcc aaccacggga tgtcctgggt ccgccgcgcg 120

cctggaaagg gcctcgaatg ggtgtcggggt attgtgtaca gcggtagcac ctactatgcc 180

gcatccgtga aggggagatt caccatcagc cgggacaact ccadgaacac tctgtacctc 240

caaatgaatt cgctgaggcc agaggacact gccatctact actgctccgc gcatggcgga 300

gagtccgacg tctggggaca ggggaccacc gtgaccgtgt ctagcgcgtc cggcggaggc 360

ggcagcgggg gtggtggttc agggggcggc ggatcggaca tccagctcac ccagtccccg 420

agctcgctgt ccgcctccgt gggagatcgg gtcaccatca cgtgccgcgc cagccagtcg 480

atttcctcct acctgaactg gtaccaacag aagcccggaa aagccccgaa gcttctcatc 540

tacgccgcct cgagcctgca gtcaggagtg ccctcacggt tctccggctc cggttccggt 600

actgatttca ccctgaccat ttcctccctg caaccggagg acttcgctac ttactactgc 660

cagcagtcgt actccacccc ctacactttc ggacaaggca ccaaggtcga aatcaag 717

<210> 202

<211> 69

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 202

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 203

<211> 207

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 203

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatcta catttgggcc cctctggctg gtacttgcgg ggtcctgctg 180

ctttcactcg tgatcactct ttactgt 207

<210> 204

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 204

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 60

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 120

gaactg 126

<210> 205

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 205

cgcgtgaaat tcagccgcag cgcagatgct ccagcctacc agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

<210> 206

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 206

Gly Ser Gly

1

<210> 207

<211> 9

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 207

ggaagcgga 9

<210> 208

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 208

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 209

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 209

gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57

<210> 210

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 210

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 211

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 211

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 212

<211> 726

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 212

gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60

ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120

ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180

aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240

gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300

ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360

ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420

tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480

cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540

taccaatcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600

tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660

tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720

tccagc 726

<210> 213

<211> 207

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 213

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 214

<211> 1000

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 214

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

165 170 175

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser

180 185 190

Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

195 200 205

Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe

210 215 220

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

225 230 235 240

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr

245 250 255

Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala

485 490 495

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

500 505 510

Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu

515 520 525

Leu Leu His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala

530 535 540

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

545 550 555 560

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

565 570 575

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

580 585 590

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

595 600 605

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

610 615 620

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

625 630 635 640

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

645 650 655

Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

660 665 670

Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp

675 680 685

Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

690 695 700

Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu

705 710 715 720

Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser

725 730 735

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

740 745 750

Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly

755 760 765

Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg

770 775 780

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

785 790 795 800

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

805 810 815

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

820 825 830

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

835 840 845

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

850 855 860

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

865 870 875 880

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

885 890 895

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

900 905 910

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

915 920 925

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

930 935 940

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

945 950 955 960

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

965 970 975

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

980 985 990

His Met Gln Ala Leu Pro Pro Arg

995 1000

<210> 215

<211> 3000

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 215

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaagtgc agttgctgga gtcaggcgga ggactggtgc agcccggagg atcgcttcgc 120

ttgagctgcg cagcctcagg ctttaccttc tcctcctacg ccatgtcctg ggtcagacag 180

gctcccggga agggactgga atgggtgtcc gccattagcg gttccggcgg aagcacttac 240

tatgccgact ctgtgaaggg ccgcttcact atctcccggg acaactccaa gaacaccctg 300

tatctccaaa tgaattccct gagggccgaa gataccgcgg tgtactactg cgctagacgg 360

gagtggtggg gagaaagctg gctgttcgac tactggggac agggcactct cgtgactgtg 420

tcctccggtg gtggtggatc ggggggtggt ggttcggggcg gaggaggatc tggagggagga 480

gggtcggaca ttcaaatgac tcagtccccg tcctccctct ccgcctccgt gggagatcgc 540

gtcacgatca cgtgcagggc cagccagagc atctccagct acctgaactg gtaccagcag 600

aagccaggga aggcaccgaa gctcctgatc tacgccgcta gctcgctgca gtccggcgtc 660

ccttcacggt tctcgggatc gggctcaggc accgacttca ccctgaccat tagcagcctg 720

cagccggagg acttcgcgac atactactgt cagcagtcat actccacccc tctgaccttc 780

ggccaaggga ccaaagtgga gatcaagacc actaccccag caccgaggcc acccaccccg 840

gctcctacca tcgcctccca gcctctgtcc ctgcgtccgg aggcatgtag acccgcagct 900

ggtggggccg tgcatacccg gggtcttgac ttcgcctgcg atatctacat ttgggcccct 960

ctggctggta cttgcggggt cctgctgctt tcactcgtga tcactcttta ctgtaagcgc 1020

ggtcggaaga agctgctgta catctttaag caacccttca tgaggcctgt gcagactact 1080

caagaggagg acggctgttc atgccggttc ccagaggagg aggaaggcgg ctgcgaactg 1140

cgcgtgaaat tcagccgcag cgcagatgct ccagcctacc agcaggggca gaaccagctc 1200

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 1260

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 1320

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 1380

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 1440

tatgacgctc ttcacatgca ggccctgccg cctcggggaa gcggagctac taacttcagc 1500

ctgctgaagc aggctggaga cgtggaggag aaccctggac ctatggcctt accagtgacc 1560

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaaat tgtgatgacc 1620

cagtcacccg ccactcttag cctttcaccc ggtgagcgcg caaccctgtc ttgcagagcc 1680

tcccaagaca tctcaaaata ccttaattgg tatcaacaga agcccggaca ggctcctcgc 1740

cttctgatct accacaccag ccggctccat tctggaatcc ctgccaggtt cagcggtagc 1800

ggatctggga ccgactacac cctcactatc agctcactgc agccagagga cttcgctgtc 1860

tatttctgtc agcaagggaa caccctgccc tacacctttg gacagggcac caagctcgag 1920

attaaaggtg gaggtggcag cggaggaggt gggtccggcg gtggaggaag ccaggtccaa 1980

ctccaagaaa gcggaccggg tcttgtgaag ccatcagaaa ctctttcact gacttgtact 2040

gtgagcggag tgtctctccc cgattacggg gtgtcttgga tcagacagcc accggggaag 2100

ggtctggaat ggattggagt gatttggggc tctgagacta cttactacca atcatccctc 2160

aagtcacgcg tcaccatctc aaaggacaac tctaagaatc aggtgtcact gaaactgtca 2220

tctgtgaccg cagccgacac cgccgtgtac tattgcgcta agcattacta ttatggcggg 2280

agctacgcaa tggattactg gggacagggt actctggtca ccgtgtccag caccacgacg 2340

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 2400

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgaggggct ggacttcgcc 2460

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 2520

gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 2580

tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 2640

gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 2700

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 2760

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 2820

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 2880

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 2940

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 3000

<210> 216

<211> 1001

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 216

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr

115 120 125

Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

145 150 155 160

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

165 170 175

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

180 185 190

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

195 200 205

Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser

210 215 220

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

225 230 235 240

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

245 250 255

Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

340 345 350

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

355 360 365

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

385 390 395 400

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

405 410 415

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

420 425 430

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

435 440 445

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

450 455 460

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly

485 490 495

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

500 505 510

Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala

515 520 525

Leu Leu Leu His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro

530 535 540

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

545 550 555 560

Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

565 570 575

Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser

580 585 590

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

595 600 605

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys

610 615 620

Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu

625 630 635 640

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

645 650 655

Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

660 665 670

Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro

675 680 685

Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

690 695 700

Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser

705 710 715 720

Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val

725 730 735

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

740 745 750

Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp

755 760 765

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro

770 775 780

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

785 790 795 800

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

805 810 815

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

820 825 830

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

835 840 845

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

850 855 860

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

865 870 875 880

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

885 890 895

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

900 905 910

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

915 920 925

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

930 935 940

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

945 950 955 960

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

965 970 975

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

980 985 990

Leu His Met Gln Ala Leu Pro Pro Arg

995 1000

<210> 217

<211> 3003

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 217

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccccaagtgc agctgcagga atccggtggc ggagtcgtgc agcctggaag gagcctgaga 120

ctctcatgcg ccgcgtcagg gttcaccttt tcctcctacg ggatgcattg ggtcagacag 180

gcccccggaa agggactcga atgggtggct gtgatcagct acgacggctc caacaagtac 240

tacgccgact ccgtgaaagg ccggttcact atctcccggg acaactccaa gaacacgctg 300

tatctgcaaa tgaattcact gcgcgcggag gataccgctg tgtactactg cggtggctcc 360

ggttacgccc tgcacgatga ctattacggc cttgacgtct ggggccaggg aaccctcgtg 420

actgtgtcca gcggtggagg aggttcgggc ggaggaggat caggaggggg tggatcgcag 480

agcgcactga ctcagccggc atccgtgtcc ggtagccccg gacagtcgat taccatctcc 540

tgtaccggca cctcctccga cgtgggaggg tacaactacg tgtcgtggta ccagcagcac 600

caggaaagg cccctaagtt gatgatctac gatgtgtcaa accgcccgtc tggagtctcc 660

aaccggttct ccggctccaa gtccggcaac accgccagcc tgaccattag cgggctgcaa 720

gccgaggatg aggccgacta ctactgctcg agctacacat cctcgagcac cctctacgtg 780

ttcggctcgg ggactaaggt caccgtgctg accactaccc cagcaccgag gccacccacc 840

ccggctccta ccatcgcctc ccagcctctg tccctgcgtc cggaggcatg tagacccgca 900

gctggtgggg ccgtgcatac ccggggtctt gacttcgcct gcgatatcta catttgggcc 960

cctctggctg gtacttgcgg ggtcctgctg ctttcactcg tgatcactct ttactgtaag 1020

cgcggtcgga agaagctgct gtacatcttt aagcaaccct tcatgaggcc tgtgcagact 1080

actcaagagg aggacggctg ttcatgccgg ttcccagagg aggaggaagg cggctgcgaa 1140

ctgcgcgtga aattcagccg cagcgcagat gctccagcct accagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcggg gaagcggagc tactaacttc 1500

agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatggc cttaccagtg 1560

accgccttgc tcctgccgct ggccttgctg ctccacgccg ccaggccgga aattgtgatg 1620

acccagtcac ccgccactct tagcctttca cccggtgagc gcgcaaccct gtcttgcaga 1680

gcctcccaag acatctcaaa ataccttaat tggtatcaac agaagcccgg acaggctcct 1740

cgccttctga tctaccacac cagccggctc cattctggaa tccctgccag gttcagcggt 1800

agcggatctg ggaccgacta caccctcact atcagctcac tgcagccaga ggacttcgct 1860

gtctatttct gtcagcaagg gaacaccctg ccctacacct ttggacaggg caccaagctc 1920

gagattaaag gtggaggtgg cagcggagga ggtgggtccg gcggtggagg aagccaggtc 1980

caactccaag aaagcggacc gggtcttgtg aagccatcag aaactctttc actgacttgt 2040

actgtgagcg gagtgtctct ccccgattac ggggtgtctt ggatcagaca gccaccgggg 2100

aagggtctgg aatggattgg agtgatttgg ggctctgaga ctacttacta ccaatcatcc 2160

ctcaagtcac gcgtcaccat ctcaaaggac aactctaaga atcaggtgtc actgaaactg 2220

tcatctgtga ccgcagccga caccgccgtg tactattgcg ctaagcatta ctattatggc 2280

gggagctacg caatggatta ctggggacag ggtactctgg tcaccgtgtc cagcaccacg 2340

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 2400

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 2460

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 2520

ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 2580

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 2640

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 2700

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 2760

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 2820

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 2880

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 2940

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 3000

cgc 3003

<210> 218

<211> 1002

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 218

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Arg Glu Trp Val Pro Tyr Asp Val Ser

115 120 125

Trp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

165 170 175

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

180 185 190

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

195 200 205

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

225 230 235 240

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr

245 250 255

Ser Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr

260 265 270

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

275 280 285

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

290 295 300

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

305 310 315 320

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

325 330 335

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

340 345 350

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

355 360 365

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

370 375 380

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

385 390 395 400

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

405 410 415

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

420 425 430

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

435 440 445

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

450 455 460

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

465 470 475 480

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser

485 490 495

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

500 505 510

Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu

515 520 525

Ala Leu Leu Leu His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser

530 535 540

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

545 550 555 560

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys

565 570 575

Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His

580 585 590

Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr

595 600 605

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe

610 615 620

Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys

625 630 635 640

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

645 650 655

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

660 665 670

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

675 680 685

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

690 695 700

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

705 710 715 720

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

725 730 735

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

740 745 750

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

755 760 765

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

770 775 780

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

785 790 795 800

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

805 810 815

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

820 825 830

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

835 840 845

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

850 855 860

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

865 870 875 880

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

885 890 895

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

900 905 910

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

915 920 925

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

930 935 940

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

945 950 955 960

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

965 970 975

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

980 985 990

Ala Leu His Met Gln Ala Leu Pro Pro Arg

995 1000

<210> 219

<211> 3006

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 219

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaagtgc agttgctgga gtcaggcgga ggactggtgc agcccggagg atcgcttcgc 120

ttgagctgcg cagcctcagg ctttaccttc tcctcctacg ccatgtcctg ggtcagacag 180

gctcccggga agggactgga atgggtgtcc gccattagcg gttccggcgg aagcacttac 240

tatgccgact ctgtgaaggg ccgcttcact atctcccggg acaactccaa gaacaccctg 300

tatctccaaa tgaattccct gagggccgaa gataccgcgg tgtactactg cgctagacgg 360

gagtgggtgc cctacgatgt cagctggtac ttcgactact ggggacaggg cactctcgtg 420

actgtgtcct ccggtggtgg tggatcgggg ggtggtggtt cgggcggagg aggatctgga 480

ggagaggagt cggacattca aatgactcag tccccgtcct ccctctccgc ctccgtggga 540

gatcgcgtca cgatcacgtg cagggccagc cagagcatct ccagctacct gaactggtac 600

cagcagaagc cagggaaggc accgaagctc ctgatctacg ccgctagctc gctgcagtcc 660

ggcgtccctt cacggttctc gggatcgggc tcaggcaccg acttcaccct gaccattagc 720

agcctgcagc cggaggactt cgcgacatac tactgtcagc agtcatactc cacccctctg 780

accttcggcc aagggaccaa agtggagatc aagaccacta ccccagcacc gaggccaccc 840

accccggctc ctaccatcgc ctcccagcct ctgtccctgc gtccggaggc atgtagaccc 900

gcagctggtg gggccgtgca tacccggggt cttgacttcg cctgcgatat ctacatttgg 960

gcccctctgg ctggtacttg cggggtcctg ctgctttcac tcgtgatcac tctttactgt 1020

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 1080

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 1140

gaactgcgcg tgaaattcag ccgcagcgca gatgctccag cctaccagca ggggcagaac 1200

cagctctaca acgaactcaa tcttggtcgg agagaggagt acgacgtgct ggacaagcgg 1260

agaggacggg acccagaaat gggcgggaag ccgcgcagaa agaatcccca agaggcctg 1320

tacaacgagc tccaaaagga taagatggca gaagcctata gcgagattgg tatgaaaggg 1380

gaacgcagaa gaggcaaagg ccacgacgga ctgtaccagg gactcagcac cgccaccaag 1440

gacacctatg acgctcttca catgcaggcc ctgccgcctc ggggaagcgg agctactaac 1500

ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggccttacca 1560

gtgaccgcct tgctcctgcc gctggccttg ctgctccacg ccgccaggcc ggaaattgtg 1620

atgacccagt cacccgccac tcttagcctt tcacccggtg agcgcgcaac cctgtcttgc 1680

agagcctccc aagacatctc aaaatacctt aattggtatc aacagaagcc cggacaggct 1740

cctcgccttc tgatctacca caccagccgg ctccattctg gaatccctgc caggttcagc 1800

ggtagcggat ctgggaccga ctacaccctc actatcagct cactgcagcc agaggacttc 1860

gctgtctatt tctgtcagca agggaacacc ctgccctaca cctttggaca gggcaccaag 1920

ctcgagatta aaggtggagg tggcagcgga ggaggtgggt ccggcggtgg aggaagccag 1980

gtccaactcc aagaaagcgg accgggtctt gtgaagccat cagaaactct ttcactgact 2040

tgtactgtga gcggagtgtc tctccccgat tacggggtgt cttggatcag acagccaccg 2100

gggaagggtc tggaatggat tggagtgatt tggggctctg agactactta ctaccaatca 2160

tccctcaagt cacgcgtcac catctcaaag gacaactcta agaatcaggt gtcactgaaa 2220

ctgtcatctg tgaccgcagc cgacaccgcc gtgtactatt gcgctaagca ttactattat 2280

ggcgggagct acgcaatgga ttactgggga cagggtactc tggtcaccgt gtccagcacc 2340

acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 2400

ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 2460

ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 2520

tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 2580

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 2640

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 2700

cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 2760

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 2820

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 2880

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 2940

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 3000

cctcgc 3006

<210> 220

<211> 991

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 220

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe

35 40 45

Ala Leu Ser Asn His Gly Met Ser Trp Val Arg Arg Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr

65 70 75 80

Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg

85 90 95

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala

100 105 110

Ile Tyr Tyr Cys Ser Ala His Gly Gly Glu Ser Asp Val Trp Gly Gln

115 120 125

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser

145 150 155 160

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

165 170 175

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys

180 185 190

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

195 200 205

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

210 215 220

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

225 230 235 240

Cys Gln Gln Ser Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys

245 250 255

Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

420 425 430

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

435 440 445

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

450 455 460

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

465 470 475 480

Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala

485 490 495

Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala

500 505 510

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Ile

515 520 525

Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

530 535 540

Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

545 550 555 560

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His

565 570 575

Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

580 585 590

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

595 600 605

Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe

610 615 620

Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly

645 650 655

Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val

660 665 670

Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro

675 680 685

Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr

690 695 700

Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp

705 710 715 720

Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala

725 730 735

Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser

740 745 750

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

755 760 765

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

770 775 780

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

785 790 795 800

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

805 810 815

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

820 825 830

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

835 840 845

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

850 855 860

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

865 870 875 880

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

885 890 895

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

900 905 910

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

915 920 925

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

930 935 940

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

945 950 955 960

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

965 970 975

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

980 985 990

<210> 221

<211> 2973

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 221

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaagtgc aattggtgga atcaggggga ggacttgtgc agcctggagg atcgctgaga 120

ctgtcatgtg ccgtgtccgg ctttgccctg tccaaccacg ggatgtcctg ggtccgccgc 180

gcgcctggaa agggcctcga atgggtgtcg ggtattgtgt acagcggtag cacctactat 240

gccgcatccg tgaaggggag attcaccatc agccgggaca actccaggaa cactctgtac 300

ctccaaatga attcgctgag gccagaggac actgccatct actactgctc cgcgcatggc 360

ggagagtccg acgtctgggg acaggggacc accgtgaccg tgtctagcgc gtccggcgga 420

ggcggcagcg ggggtggtgg ttcaggggggc ggcggatcgg acatccagct cacccagtcc 480

ccgagctcgc tgtccgcctc cgtgggagat cgggtcacca tcacgtgccg cgccagccag 540

tcgatttcct cctacctgaa ctggtaccaa cagaagcccg gaaaagcccc gaagcttctc 600

atctacgccg cctcgagcct gcagtcagga gtgccctcac ggttctccgg ctccggttcc 660

ggtactgatt tcaccctgac catttcctcc ctgcaaccgg aggacttcgc tacttactac 720

tgccagcagt cgtactccac cccctacact ttcggacaag gcaccaaggt cgaaatcaag 780

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 840

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 900

gacttcgcct gcgatatcta catttggggcc cctctggctg gtacttgcgg ggtcctgctg 960

ctttcactcg tgatcactct ttactgtaag cgcggtcgga agaagctgct gtacatcttt 1020

aagcaaccct tcatgaggcc tgtgcagact actcaagagg aggacggctg ttcatgccgg 1080

ttcccagagg aggaggaagg cggctgcgaa ctgcgcgtga aattcagccg cagcgcagat 1140

gctccagcct accagcaggg gcagaaccag ctctacaacg aactcaatct tggtcggaga 1200

gaggagtacg acgtgctgga caagcggaga ggacgggacc cagaaatggg cgggaagccg 1260

cgcagaaaga atccccaaga gggcctgtac aacgagctcc aaaaggataa gatggcagaa 1320

gcctatagcg agattggtat gaaaggggaa cgcagaagag gcaaaggcca cgacggactg 1380

taccagggac tcagcaccgc caccaaggac acctatgacg ctcttcacat gcaggccctg 1440

ccgcctcggg gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag 1500

gagaaccctg gacctatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 1560

ctccacgccg ccaggccgga aattgtgatg acccagtcac ccgccactct tagcctttca 1620

cccggtgagc gcgcaaccct gtcttgcaga gcctcccaag acatctcaaa ataccttaat 1680

tggtatcaac agaagcccgg acaggctcct cgccttctga tctaccacac cagccggctc 1740

cattctggaa tccctgccag gttcagcggt agcggatctg ggaccgacta caccctcact 1800

atcagctcac tgcagccaga ggacttcgct gtctatttct gtcagcaagg gaacaccctg 1860

ccctacacct ttggacaggg caccaagctc gagattaaag gtggaggtgg cagcggagga 1920

ggtgggtccg gcggtggagg aagccaggtc caactccaag aaagcggacc gggtcttgtg 1980

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac 2040

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg 2100

ggctctgaga ctacttacta ccaatcatcc ctcaagtcac gcgtcaccat ctcaaaggac 2160

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg 2220

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag 2280

ggtactctgg tcaccgtgtc cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 2340

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 2400

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 2460

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 2520

agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 2580

gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 2640

gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 2700

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 2760

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 2820

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 2880

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 2940

gacgcccttc acatgcaggc cctgccccct cgc 2973

<210> 222

<211> 991

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 222

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro

500 505 510

Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg

515 520 525

Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

530 535 540

Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ala Leu Ser Asn

545 550 555 560

His Gly Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp

565 570 575

Val Ser Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr Ala Ala Ser Val

580 585 590

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr

595 600 605

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys

610 615 620

Ser Ala His Gly Gly Glu Ser Asp Val Trp Gly Gln Gly Thr Thr Val

625 630 635 640

Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

645 650 655

Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu

660 665 670

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln

675 680 685

Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

690 695 700

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

705 710 715 720

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

725 730 735

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser

740 745 750

Tyr Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

755 760 765

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

770 775 780

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

785 790 795 800

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

805 810 815

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

820 825 830

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

835 840 845

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

850 855 860

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

865 870 875 880

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

885 890 895

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

900 905 910

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

915 920 925

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

930 935 940

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

945 950 955 960

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

965 970 975

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

980 985 990

<210> 223

<211> 2973

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 223

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca 120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag 180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct 240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag 300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga 360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt 420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact 480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc 540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact 600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag 660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag 720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc 780

gtgtccagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

aggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg aagcggagct actaacttca gcctgctgaa gcaggctgga 1500

gacgtggagg agaaccctgg acctatggcc ctccctgtca ccgccctgct gcttccgctg 1560

gctcttctgc tccacgccgc tcggcccgaa gtgcaattgg tggaatcagg gggaggactt 1620

gtgcagcctg gaggatcgct gagactgtca tgtgccgtgt ccggctttgc cctgtccaac 1680

cacgggatgt cctgggtccg ccgcgcgcct ggaaagggcc tcgaatgggt gtcgggtatt 1740

gtgtacagcg gtagcaccta ctatgccgca tccgtgaagg ggagattcac catcagccgg 1800

gacaactcca ggaacactct gtacctccaa atgaattcgc tgaggccaga ggacactgcc 1860

atctactact gctccgcgca tggcggagag tccgacgtct ggggacaggg gaccaccgtg 1920

accgtgtcta gcgcgtccgg cggaggcggc agcgggggtg gtggttcagg gggcggcgga 1980

tcggacatcc agctcaccca gtccccgagc tcgctgtccg cctccgtggg agatcgggtc 2040

accatcacgt gccgcgccag ccagtcgatt tcctcctacc tgaactggta ccaacagaag 2100

cccggaaaag ccccgaagct tctcatctac gccgcctcga gcctgcagtc aggagtgccc 2160

tcacggttct ccggctccgg ttccggtact gatttcaccc tgaccatttc ctccctgcaa 2220

ccggaggact tcgctactta ctactgccag cagtcgtact ccacccccta cactttcgga 2280

caaggcacca aggtcgaaat caagaccact accccagcac cgaggccacc caccccggct 2340

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt 2400

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg 2460

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt 2520

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 2580

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc 2640

gtgaaattca gccgcagcgc agatgctcca gcctaccagc aggggcagaa ccagctctac 2700

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg 2760

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag 2820

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga 2880

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat 2940

gacgctcttc acatgcaggc cctgccgcct cgg 2973

<210> 224

<211> 492

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 224

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

180 185 190

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

210 215 220

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

225 230 235 240

Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

245 250 255

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

290 295 300

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

305 310 315 320

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

325 330 335

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

340 345 350

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

355 360 365

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

370 375 380

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

450 455 460

Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu

465 470 475 480

Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

485 490

<210> 225

<211> 465

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 225

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

245 250 255

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

260 265 270

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

275 280 285

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

290 295 300

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

305 310 315 320

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

325 330 335

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

340 345 350

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 226

<211> 493

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 226

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln

130 135 140

Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys

145 150 155 160

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr

165 170 175

Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser

180 185 190

Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly

195 200 205

Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala

210 215 220

Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr Val Phe

225 230 235 240

Gly Ser Gly Thr Lys Val Thr Val Leu Thr Thr Thr Pro Ala Pro Arg

245 250 255

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

260 265 270

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

275 280 285

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

290 295 300

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

305 310 315 320

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

325 330 335

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

340 345 350

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

355 360 365

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

370 375 380

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

385 390 395 400

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

405 410 415

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

420 425 430

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

435 440 445

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

450 455 460

His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser

465 470 475 480

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

485 490

<210> 227

<211> 494

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 227

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Val Pro Tyr Asp Val Ser Trp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

130 135 140

Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

145 150 155 160

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu

165 170 175

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

180 185 190

Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

210 215 220

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr

225 230 235 240

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro

245 250 255

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

260 265 270

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

275 280 285

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

290 295 300

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys

305 310 315 320

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

325 330 335

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

340 345 350

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

355 360 365

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

370 375 380

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

385 390 395 400

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

405 410 415

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

420 425 430

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

435 440 445

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

450 455 460

Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe

465 470 475 480

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

485 490

<210> 228

<211> 483

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 228

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ala Leu Ser Asn His

20 25 30

Gly Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr Ala Ala Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser

85 90 95

Ala His Gly Gly Glu Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr

210 215 220

Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr

225 230 235 240

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

245 250 255

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

260 265 270

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

275 280 285

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

290 295 300

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

305 310 315 320

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

325 330 335

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

340 345 350

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

355 360 365

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

370 375 380

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

385 390 395 400

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

405 410 415

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

420 425 430

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

435 440 445

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser

450 455 460

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

465 470 475 480

Asn Pro Gly

<210> 229

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 229

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

245 250 255

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

260 265 270

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

275 280 285

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

290 295 300

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

305 310 315 320

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

325 330 335

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

340 345 350

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp

465 470 475 480

Val Glu Glu Asn Pro Gly

485

<210> 230

<211> 462

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 230

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ala Leu Ser Asn His

20 25 30

Gly Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr Ala Ala Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser

85 90 95

Ala His Gly Gly Glu Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr

210 215 220

Ser Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr

225 230 235 240

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

245 250 255

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

260 265 270

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

275 280 285

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

290 295 300

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

305 310 315 320

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

325 330 335

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

340 345 350

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

355 360 365

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

370 375 380

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

385 390 395 400

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

405 410 415

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

420 425 430

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

435 440 445

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

450 455 460

<210> 231

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 231

Asn His Gly Met Ser

1 5

<210> 232

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 232

Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr Ala Ala Ser Val Lys Gly

1 5 10 15

<210> 233

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 233

His Gly Gly Glu Ser Asp Val

1 5

<210> 234

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 234

Gly Phe Ala Leu Ser Asn His

1 5

<210> 235

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 235

Val Tyr Ser Gly Ser

1 5

<210> 236

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 236

Gly Phe Ala Leu Ser Asn His Gly

1 5

<210> 237

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 237

Ile Val Tyr Ser Gly Ser Thr

1 5

<210> 238

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 238

Ser Ala His Gly Gly Glu Ser Asp Val

1 5

<210> 239

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 239

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ala Leu Ser Asn His

20 25 30

Gly Met Ser Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Val Tyr Ser Gly Ser Thr Tyr Tyr Ala Ala Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ser

85 90 95

Ala His Gly Gly Glu Ser Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 240

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 240

Gln Gln Ser Tyr Ser Thr Pro Tyr Thr

1 5

<210> 241

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 241

Ser Tyr Ser Thr Pro Tyr

1 5

<210> 242

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 242

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 243

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 243

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10 15

Ser

<210> 244

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 244

Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

1 5 10

<210> 245

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 245

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

1 5 10 15

<210> 246

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 246

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 247

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 247

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 248

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 248

His Thr Ser Arg Leu His Ser

1 5

<210> 249

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 249

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 250

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 250

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 251

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 251

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 252

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 252

atggccctcc ctgtcaccgc tctgttgctg ccgcttgctc tgctgctcca cgcagcgcga 60

ccg 63

<210> 253

<211> 747

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 253

caggtacaat tgcaggagtc tggaggcggt gtggtgcaac ccggtcgcag cttgcgcctg 60

agttgtgctg cgtctggatt tacattttca tcttacggaa tgcattgggt acgccaggca 120

ccggggaaag gccttgaatg ggtggctgta atttcatacg atggttccaa caaatactat 180

gctgactcag tcaagggtcg atttacaatt agtcgggaca actccaagaa caccctttat 240

cttcaaatga attcccttag agcagaggat acggcggtct attactgtgg tggcagtggt 300

tatgcacttc atgatgatta ctatggcttg gatgtctggg ggcaagggac gcttgtaact 360

gtatcctctg gtggtggtgg tagtggtggg ggaggctccg gcggtggcgg ctctcaatct 420

gctctgactc aaccagcaag cgtatcaggg tcaccgggac agagtattac cataagttgc 480

acggggacct ctagcgatgt aggggggtat aattatgtat cttggtatca acaacacccc 540

gggaaagccc ctaaattgat gatctacgac gtgagcaatc gacctagtgg cgtatcaaat 600

cgcttctctg gtagcaagag tgggaatacg gcgtccctta ctattagcgg attgcaagca 660

gaagatgagg ccgattacta ctgcagctcc tatactagct cttctacatt gtacgtcttt 720

gggagcggaa caaaagtaac agtactc 747

<210> 254

<211> 207

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 254

acaacaacac ctgccccgag accgcctaca ccagccccga ctattgccag ccagcctctg 60

agcctcaggc ctgaggcctg taggcccgca gcgggcggcg cagttcatac acggggcttg 120

gatttcgctt gtgatattta tatttgggct cctttggcgg ggacatgtgg cgtgctgctt 180

ctgtcacttg ttattacact gtactgt 207

<210> 255

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 255

aaacgcgggc gaaaaaaatt gctgtatatt tttaagcagc catttatgag gcccgttcag 60

acgacgcagg aggaggacgg ttgctcttgc aggttcccag aagaggaaga aggggctgt 120

gaattg 126

<210> 256

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 256

cgggttaaat tttcaagatc cgcagacgct ccagcatacc aacagggaca aaaccaactc 60

tataacgagc tgaatcttgg aagaagggag gaatatgatg tgctggataa acggcgcggt 120

agagatccgg agatgggcgg aaaaccaagg cgaaaaaacc ctcaggaggg actctacaac 180

gaactgcaga aagacaaaat ggcggaggct tattccgaaa taggcatgaa gggcgagcgg 240

aggcgaggga aagggcacga cggactgtat caaggcctct caaccgcgac taaggatacg 300

tacgacgccc tgcacatgca ggccctgcct ccgaga 336

<210> 257

<211> 493

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 257

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr

115 120 125

Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

145 150 155 160

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

165 170 175

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

180 185 190

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

195 200 205

Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser

210 215 220

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

225 230 235 240

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

245 250 255

Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

340 345 350

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

355 360 365

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

385 390 395 400

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

405 410 415

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

420 425 430

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

435 440 445

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

450 455 460

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 258

<211> 1479

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 258

atggccctcc ctgtcaccgc tctgttgctg ccgcttgctc tgctgctcca cgcagcgcga 60

ccgcaggtac aattgcagga gtctggaggc ggtgtggtgc aacccggtcg cagcttgcgc 120

ctgagttgtg ctgcgtctgg atttacattt tcatcttacg gaatgcattg ggtacgccag 180

gcaccgggga aaggccttga atgggtggct gtaatttcat acgatggttc caacaaatac 240

tatgctgact cagtcaaggg tcgatttaca attagtcggg acaactccaa gaacaccctt 300

tatcttcaaa tgaattccct tagagcagag gatacggcgg tctattactg tggtggcagt 360

ggttatgcac ttcatgatga ttactatggc ttggatgtct gggggcaagg gacgcttgta 420

actgtatcct ctggtggtgg tggtagtggt gggggaggct ccggcggtgg cggctctcaa 480

tctgctctga ctcaaccagc aagcgtatca gggtcaccgg gacagagtat taccataagt 540

tgcacgggga cctctagcga tgtagggggg tataattatg tatcttggta tcaacaacac 600

cccgggaaag cccctaaatt gatgatctac gacgtgagca atcgacctag tggcgtatca 660

aatcgcttct ctggtagcaa gagtgggaat acggcgtccc ttactattag cggattgcaa 720

gcagaagatg aggccgatta ctactgcagc tcctatacta gctcttctac attgtacgtc 780

tttgggagcg gaacaaaagt aacagtactc acaacaacac ctgccccgag accgcctaca 840

ccagccccga ctattgccag ccagcctctg agcctcaggc ctgaggcctg taggcccgca 900

gcgggcggcg cagttcatac acggggcttg gatttcgctt gtgatattta tatttgggct 960

cctttggcgg ggacatgtgg cgtgctgctt ctgtcacttg ttattacact gtactgtaaa 1020

cgcgggcgaa aaaaattgct gtatattttt aagcagccat ttatgaggcc cgttcagacg 1080

acgcaggagg aggacggttg ctcttgcagg ttcccagaag aggaagaagg gggctgtgaa 1140

ttgcgggtta aattttcaag atccgcagac gctccagcat accaacaggg acaaaaccaa 1200

ctctataacg agctgaatct tggaagaagg gaggaatatg atgtgctgga taaacggcgc 1260

ggtagagatc cggagatggg cggaaaacca aggcgaaaaa accctcagga gggactctac 1320

aacgaactgc agaaagacaa aatggcggag gcttattccg aaataggcat gaagggcgag 1380

cggaggcgag ggaaagggca cgacggactg tatcaaggcc tctcaaccgc gactaaggat 1440

acgtacgacg ccctgcacat gcaggccctg cctccgaga 1479

<210> 259

<211> 1416

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 259

caggtacaat tgcaggagtc tggaggcggt gtggtgcaac ccggtcgcag cttgcgcctg 60

agttgtgctg cgtctggatt tacattttca tcttacggaa tgcattgggt acgccaggca 120

ccggggaaag gccttgaatg ggtggctgta atttcatacg atggttccaa caaatactat 180

gctgactcag tcaagggtcg atttacaatt agtcgggaca actccaagaa caccctttat 240

cttcaaatga attcccttag agcagaggat acggcggtct attactgtgg tggcagtggt 300

tatgcacttc atgatgatta ctatggcttg gatgtctggg ggcaagggac gcttgtaact 360

gtatcctctg gtggtggtgg tagtggtggg ggaggctccg gcggtggcgg ctctcaatct 420

gctctgactc aaccagcaag cgtatcaggg tcaccgggac agagtattac cataagttgc 480

acggggacct ctagcgatgt aggggggtat aattatgtat cttggtatca acaacacccc 540

gggaaagccc ctaaattgat gatctacgac gtgagcaatc gacctagtgg cgtatcaaat 600

cgcttctctg gtagcaagag tgggaatacg gcgtccctta ctattagcgg attgcaagca 660

gaagatgagg ccgattacta ctgcagctcc tatactagct cttctacatt gtacgtcttt 720

gggagcggaa caaaagtaac agtactcaca acaacacctg ccccgagacc gcctacacca 780

gccccgacta ttgccagcca gcctctgagc ctcaggcctg aggcctgtag gcccgcagcg 840

ggcggcgcag ttcatacacg gggcttggat ttcgcttgtg atatttatat ttgggctcct 900

ttggcgggga catgtggcgt gctgcttctg tcacttgtta ttacactgta ctgtaaacgc 960

gggcgaaaaa aattgctgta tatttttaag cagccattta tgaggcccgt tcagacgacg 1020

caggaggagg acggttgctc ttgcaggttc ccagaagagg aagaaggggg ctgtgaattg 1080

cgggttaaat tttcaagatc cgcagacgct ccagcatacc aacagggaca aaaccaactc 1140

tataacgagc tgaatcttgg aagaagggag gaatatgatg tgctggataa acggcgcggt 1200

agagatccgg agatgggcgg aaaaccaagg cgaaaaaacc ctcaggaggg actctacaac 1260

gaactgcaga aagacaaaat ggcggaggct tattccgaaa taggcatgaa gggcgagcgg 1320

aggcgaggga aagggcacga cggactgtat caaggcctct caaccgcgac taaggatacg 1380

tacgacgccc tgcacatgca ggccctgcct ccgaga 1416

<210> 260

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 260

caggtacaat tgcaggagtc tggaggcggt gtggtgcaac ccggtcgcag cttgcgcctg 60

agttgtgctg cgtctggatt tacattttca tcttacggaa tgcattgggt acgccaggca 120

ccggggaaag gccttgaatg ggtggctgta atttcatacg atggttccaa caaatactat 180

gctgactcag tcaagggtcg atttacaatt agtcgggaca actccaagaa caccctttat 240

cttcaaatga attcccttag agcagaggat acggcggtct attactgtgg tggcagtggt 300

tatgcacttc atgatgatta ctatggcttg gatgtctggg ggcaagggac gcttgtaact 360

gtaccctct 369

<210> 261

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 261

caatctgctc tgactcaacc agcaagcgta tcagggtcac cgggacagag tattaccata 60

agttgcacgg ggacctctag cgatgtaggg gggtataatt atgtatcttg gtatcaacaa 120

caccccggga aagcccctaa attgatgatc tacgacgtga gcaatcgacc tagtggcgta 180

tcaaatcgct tctctggtag caagagtggg aatacggcgt cccttactat tagcggattg 240

caagcagaag atgaggccga ttactactgc agctcctata ctagctcttc tacattgtac 300

gtctttggga gcggaacaaa agtaacagta ctc 333

<210> 262

<211> 345

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 262

gaagtgcaat tggtggaatc agggggagga cttgtgcagc ctggaggatc gctgagactg 60

tcatgtgccg tgtccggctt tgccctgtcc aaccacggga tgtcctgggt ccgccgcgcg 120

cctggaaagg gcctcgaatg ggtgtcggggt attgtgtaca gcggtagcac ctactatgcc 180

gcatccgtga aggggagatt caccatcagc cgggacaact ccadgaacac tctgtacctc 240

caaatgaatt cgctgaggcc agaggacact gccatctact actgctccgc gcatggcgga 300

gagtccgacg tctggggaca ggggaccacc gtgaccgtgt ctagc 345

<210> 263

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 263

gacatccagc tcacccagtc cccgagctcg ctgtccgcct ccgtgggaga tcgggtcacc 60

atcacgtgcc gcgccagcca gtcgatttcc tcctacctga actggtacca acagaagccc 120

ggaaaagccc cgaagcttct catctacgcc gcctcgagcc tgcagtcagg agtgccctca 180

cggttctccg gctccggttc cggtactgat ttcaccctga ccatttcctc cctgcaaccg 240

gaggacttcg ctacttacta ctgccagcag tcgtactcca ccccctacac tttcggacaa 300

ggcaccaagg tcgaaatcaa g 321

<210> 264

<400> 264

000

<210> 265

<400> 265

000

<210> 266

<400> 266

000

<210> 267

<400> 267

000

<210> 268

<400> 268

000

<210> 269

<400> 269

000

<210> 270

<400> 270

000

<210> 271

<400> 271

000

<210> 272

<400> 272

000

<210> 273

<400> 273

000

<210> 274

<400> 274

000

<210> 275

<211> 132

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 275

Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly Pro Ser Ser Pro Lys Lys

1 5 10 15

Lys Arg Lys Val Ser Arg Gly Val Gln Val Glu Thr Ile Ser Pro Gly

20 25 30

Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr

35 40 45

Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg

50 55 60

Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly

65 70 75 80

Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu

85 90 95

Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile

100 105 110

Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu

115 120 125

Glu Thr Ser Tyr

130

<210> 276

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 276

Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys

1 5 10 15

Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly

20 25 30

Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met

35 40 45

Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln

50 55 60

Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala

65 70 75 80

Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu

85 90 95

Val Phe Asp Val Glu Leu Leu Lys Leu Glu Thr Ser

100 105

<210> 277

<211> 93

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 277

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys

85 90

<210> 278

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 278

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 279

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 279

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 280

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 280

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 281

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<220>

<221>MOD_RES

<222> (12)..(12)

<223> Any amino acid

<220>

<221>MOD_RES

<222> (78)..(78)

<223> Any amino acid

<400> 281

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Xaa Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Xaa Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 282

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 282

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 283

<211> 95

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 283

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 284

<211> 1132

<212> PRT

<213> Homo sapiens

<400> 284

Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro

340 345 350

Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Gly Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro

690 695 700

Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile

705 710 715 720

Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys

885 890 895

Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu

900 905 910

Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe

945 950 955 960

Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln

1010 1015 1020

Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp

1025 1030 1035

Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly

1040 1045 1050

Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu

1055 1060 1065

Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr

1070 1075 1080

Arg His Arg Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr

1085 1090 1095

Ala Gln Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr

1100 1105 1110

Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys

1115 1120 1125

Th Ile Leu Asp

1130

<210> 285

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 285

Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Met Leu Ser Asn

20 25 30

Ser Asp Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr His Arg Ser Thr Trp Tyr Asp Asp Tyr Ala

50 55 60

Ser Ser Val Arg Gly Arg Val Ser Ile Asn Val Asp Thr Ser Lys Asn

65 70 75 80

Gln Tyr Ser Leu Gln Leu Asn Ala Val Thr Pro Glu Asp Thr Gly Val

85 90 95

Tyr Tyr Cys Ala Arg Val Arg Leu Gln Asp Gly Asn Ser Trp Ser Asp

100 105 110

Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser

130 135 140

Ala Leu Thr Gln Pro Ala Ser Ala Ser Gly Ser Pro Gly Gln Ser Val

145 150 155 160

Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr

165 170 175

Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile

180 185 190

Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly

195 200 205

Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala

210 215 220

Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr

225 230 235 240

Leu Tyr Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu

245 250

<210> 286

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 286

Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Met Leu Ser Asn

20 25 30

Ser Asp Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr His Arg Ser Thr Trp Tyr Asp Asp Tyr Ala

50 55 60

Ser Ser Val Arg Gly Arg Val Ser Ile Asn Val Asp Thr Ser Lys Asn

65 70 75 80

Gln Tyr Ser Leu Gln Leu Asn Ala Val Thr Pro Glu Asp Thr Gly Val

85 90 95

Tyr Tyr Cys Ala Arg Val Arg Leu Gln Asp Gly Asn Ser Trp Ser Asp

100 105 110

Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 287

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 287

Gln Ser Ala Leu Thr Gln Pro Ala Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu

100 105 110

<210> 288

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 288

Gly Asp Ser Met Leu Ser Asn Ser Asp Thr Trp Asn

1 5 10

<210> 289

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 289

Ser Asn Ser Asp Thr Trp Asn

1 5

<210> 290

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 290

Arg Thr Tyr His Arg Ser Thr Trp Tyr Asp Asp Tyr Ala Ser Ser Val

1 5 10 15

Arg Gly

<210> 291

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 291

Val Arg Leu Gln Asp Gly Asn Ser Trp Ser Asp Ala Phe Asp Val

1 5 10 15

<210> 292

<211> 465

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 292

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

245 250 255

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

260 265 270

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

275 280 285

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

290 295 300

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

305 310 315 320

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

325 330 335

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

340 345 350

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 293

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 293

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 294

<211> 465

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 294

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

245 250 255

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

260 265 270

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

275 280 285

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

290 295 300

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

305 310 315 320

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

325 330 335

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

340 345 350

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 295

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 295

Asp Tyr Gly Val Ser

1 5

<210> 296

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 296

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 297

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 297

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 298

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 298

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 299

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 299

His Thr Ser Arg Leu His Ser

1 5

<210> 300

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 300

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 301

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 301

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 302

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 302

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

aggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgc 1458

<210> 303

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 303

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 304

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 304

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

1 5 10 15

<210> 305

<211> 726

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 305

gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60

ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120

ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180

aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240

gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300

ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360

ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420

tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480

cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540

taccaatcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600

tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660

tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720

tccagc 726

<210> 306

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 306

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 307

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 307

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca 120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag 180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct 240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag 300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga 360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt 420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact 480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc 540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact 600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag 660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag 720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc 780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc 840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc 900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg 960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg 1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt 1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc 1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt 1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag 1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac 1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg 1440

caggccctgc cgcctcgg 1458

<210> 308

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 308

Leu Ala Glu Ala Ala Ala Lys

1 5

<210> 309

<211> 284

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 309

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

115 120 125

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

130 135 140

Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val

145 150 155 160

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

165 170 175

Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser

180 185 190

Thr Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro

195 200 205

Ser Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala

210 215 220

Ala Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys

225 230 235 240

Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His

245 250 255

Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser

260 265 270

Ala Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly

275 280

<210> 310

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 310

Gly Val Ser Leu Pro Asp Tyr

1 5

<210> 311

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 311

TrpGlySerGluThr

1 5

<210> 312

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 312

Gly Val Ser Leu Pro Asp Tyr Gly

1 5

<210> 313

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 313

Ile Trp Gly Ser Glu Thr Thr

1 5

<210> 314

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 314

Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 315

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 315

caggtccaac tccaagaaag cggaccgggt cttgtgaagc catcagaaac tctttcactg 60

acttgtactg tgagcggagt gtctctcccc gattacgggg tgtcttggat cagacagcca 120

ccggggaagg gtctggaatg gattggagtg atttggggct ctgagactac ttactaccaa 180

tcatccctca agtcacgcgt caccatctca aaggacaact ctaagaatca ggtgtcactg 240

aaactgtcat ctgtgaccgc agccgacacc gccgtgtact attgcgctaa gcattactat 300

tatggcggga gctacgcaat ggattactgg ggacagggta ctctggtcac cgtgtccagc 360

<210> 316

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 316

Ser Gln Asp Ile Ser Lys Tyr

1 5

<210> 317

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 317

His Thr Ser

1

<210> 318

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 318

Gly Asn Thr Leu Pro Tyr

1 5

<210> 319

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 319

Gln Asp Ile Ser Lys Tyr

1 5

<210> 320

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 320

gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60

ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120

ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180

aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240

gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300

ggcaccaagc tcgagattaa a 321

<210> 321

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 321

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Ser Ala

115 120 125

Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr

130 135 140

Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val

145 150 155 160

Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

165 170 175

Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser

180 185 190

Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu

195 200 205

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu

210 215 220

Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

260 265 270

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

275 280 285

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

290 295 300

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

305 310 315 320

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

325 330 335

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

340 345 350

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

355 360 365

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

370 375 380

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

385 390 395 400

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

405 410 415

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

420 425 430

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

435 440 445

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

450 455 460

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

465 470 475 480

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

485 490

<210> 322

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 322

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

115 120 125

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

130 135 140

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

145 150 155 160

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

165 170 175

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

180 185 190

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

195 200 205

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

210 215 220

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

245 250 255

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

260 265 270

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

275 280 285

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

290 295 300

Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser

305 310 315 320

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

325 330 335

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

340 345 350

Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gly

355 360 365

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

370 375 380

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

385 390 395 400

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu

405 410 415

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

420 425 430

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

435 440 445

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

450 455 460

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

485 490

<210> 323

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 323

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln

115 120 125

Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

165 170 175

Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Gly Ser

210 215 220

Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu

260 265 270

Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile

275 280 285

Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

290 295 300

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp

305 310 315 320

Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys

325 330 335

Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp

340 345 350

Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr

355 360 365

Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly Ser

370 375 380

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

385 390 395 400

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

405 410 415

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

420 425 430

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

435 440 445

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

450 455 460

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

465 470 475 480

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

485 490

<210> 324

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 324

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

115 120 125

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

130 135 140

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

145 150 155 160

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

165 170 175

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

180 185 190

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

195 200 205

Ser Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln

245 250 255

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

260 265 270

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

275 280 285

Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

290 295 300

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

305 310 315 320

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

325 330 335

Tyr Tyr Cys Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly

340 345 350

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

370 375 380

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

385 390 395 400

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu

405 410 415

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

420 425 430

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

435 440 445

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

450 455 460

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

485 490

<210> 325

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 325

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

145 150 155 160

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

165 170 175

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

195 200 205

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

210 215 220

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

245 250 255

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

260 265 270

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

275 280 285

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

290 295 300

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

305 310 315 320

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

325 330 335

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

340 345 350

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu

370 375 380

Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile

385 390 395 400

Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

405 410 415

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp

420 425 430

Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys

435 440 445

Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp

450 455 460

Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr

465 470 475 480

Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu

485 490

<210> 326

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 326

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Gly

100 105 110

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

115 120 125

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

130 135 140

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

145 150 155 160

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

165 170 175

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

180 185 190

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

195 200 205

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

210 215 220

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

260 265 270

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

275 280 285

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

290 295 300

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

305 310 315 320

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

325 330 335

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

340 345 350

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

355 360 365

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

370 375 380

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

385 390 395 400

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

405 410 415

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

420 425 430

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

435 440 445

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

450 455 460

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

485 490

<210> 327

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 327

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

130 135 140

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

145 150 155 160

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

165 170 175

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

180 185 190

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

195 200 205

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

260 265 270

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

275 280 285

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

290 295 300

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

305 310 315 320

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

325 330 335

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

340 345 350

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

355 360 365

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Ser Ala Leu

370 375 380

Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile

385 390 395 400

Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

405 410 415

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp

420 425 430

Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys

435 440 445

Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp

450 455 460

Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr

465 470 475 480

Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu

485 490

<210> 328

<211> 491

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 328

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Gly

100 105 110

Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser

115 120 125

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp

130 135 140

Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

145 150 155 160

Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala

165 170 175

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser

180 185 190

Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn

195 200 205

Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

245 250 255

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

260 265 270

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

275 280 285

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

290 295 300

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

305 310 315 320

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

325 330 335

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

340 345 350

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

355 360 365

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

370 375 380

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

385 390 395 400

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

405 410 415

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

420 425 430

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

435 440 445

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

450 455 460

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

485 490

<210> 329

<211> 485

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 329

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln

115 120 125

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

130 135 140

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

145 150 155 160

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

165 170 175

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

180 185 190

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

195 200 205

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

210 215 220

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

245 250 255

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

260 265 270

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp

275 280 285

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser

290 295 300

Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

305 310 315 320

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

325 330 335

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Glu

340 345 350

Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln

370 375 380

Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser

385 390 395 400

Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln

405 410 415

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu

420 425 430

His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

435 440 445

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr

450 455 460

Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Cys Gly Thr

465 470 475 480

Lys Leu Glu Ile Lys

485

<210> 330

<211> 485

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 330

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile

115 120 125

Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

130 135 140

Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

145 150 155 160

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His

165 170 175

Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

180 185 190

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

195 200 205

Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe

210 215 220

Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

245 250 255

Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser

260 265 270

Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

275 280 285

Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile

290 295 300

Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val

305 310 315 320

Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser

325 330 335

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr

340 345 350

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

370 375 380

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

385 390 395 400

Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln

405 410 415

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu

420 425 430

Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

435 440 445

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

450 455 460

Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Cys Gly Thr

465 470 475 480

Lys Val Glu Ile Lys

485

<210> 331

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 331

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 332

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 332

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 333

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 333

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 334

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 334

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 335

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 335

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 336

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 336

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

100 105

<210> 337

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 337

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgat 135

<210> 338

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 338

atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60

actctttact gt 72

<210> 339

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 339

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Ser Ala

115 120 125

Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr

130 135 140

Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val

145 150 155 160

Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

165 170 175

Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser

180 185 190

Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu

195 200 205

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu

210 215 220

Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

260 265 270

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

275 280 285

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

290 295 300

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

305 310 315 320

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

325 330 335

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

340 345 350

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

355 360 365

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

370 375 380

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

385 390 395 400

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

405 410 415

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

420 425 430

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

435 440 445

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

450 455 460

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

465 470 475 480

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 340

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 340

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

115 120 125

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

130 135 140

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

145 150 155 160

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

165 170 175

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

180 185 190

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

195 200 205

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

210 215 220

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

245 250 255

Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser

260 265 270

Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

275 280 285

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

290 295 300

Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser

305 310 315 320

Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln

325 330 335

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

340 345 350

Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gly

355 360 365

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

370 375 380

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

385 390 395 400

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu

405 410 415

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

420 425 430

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

435 440 445

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

450 455 460

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 341

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 341

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Val Gln

115 120 125

Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

165 170 175

Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Gly Ser

210 215 220

Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu

260 265 270

Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile

275 280 285

Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

290 295 300

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp

305 310 315 320

Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys

325 330 335

Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp

340 345 350

Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr

355 360 365

Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly Ser

370 375 380

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

385 390 395 400

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

405 410 415

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

420 425 430

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

435 440 445

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

450 455 460

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

465 470 475 480

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 342

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 342

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

115 120 125

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

130 135 140

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

145 150 155 160

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

165 170 175

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

180 185 190

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

195 200 205

Ser Thr Leu Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln

245 250 255

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

260 265 270

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

275 280 285

Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

290 295 300

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

305 310 315 320

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

325 330 335

Tyr Tyr Cys Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly

340 345 350

Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

370 375 380

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

385 390 395 400

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu

405 410 415

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

420 425 430

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

435 440 445

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

450 455 460

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 343

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 343

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

145 150 155 160

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

165 170 175

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

195 200 205

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

210 215 220

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

245 250 255

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

260 265 270

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

275 280 285

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

290 295 300

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

305 310 315 320

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

325 330 335

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

340 345 350

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu

370 375 380

Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile

385 390 395 400

Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

405 410 415

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp

420 425 430

Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys

435 440 445

Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp

450 455 460

Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr

465 470 475 480

Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 344

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 344

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Gly

100 105 110

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

115 120 125

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

130 135 140

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

145 150 155 160

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

165 170 175

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

180 185 190

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

195 200 205

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

210 215 220

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

260 265 270

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

275 280 285

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

290 295 300

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

305 310 315 320

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

325 330 335

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

340 345 350

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

355 360 365

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

370 375 380

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

385 390 395 400

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

405 410 415

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

420 425 430

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

435 440 445

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

450 455 460

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 345

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 345

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

130 135 140

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

145 150 155 160

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

165 170 175

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

180 185 190

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

195 200 205

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

260 265 270

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

275 280 285

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

290 295 300

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

305 310 315 320

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

325 330 335

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

340 345 350

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

355 360 365

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gln Ser Ala Leu

370 375 380

Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile

385 390 395 400

Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

405 410 415

Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp

420 425 430

Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys

435 440 445

Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp

450 455 460

Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Leu Tyr

465 470 475 480

Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 346

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 346

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Tyr Val Phe Gly Cys Gly Thr Lys Val Thr Val Leu Gly

100 105 110

Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser

115 120 125

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp

130 135 140

Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

145 150 155 160

Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala

165 170 175

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser

180 185 190

Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn

195 200 205

Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys

245 250 255

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

260 265 270

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

275 280 285

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser

290 295 300

Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln

305 310 315 320

Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

325 330 335

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

340 345 350

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

355 360 365

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

370 375 380

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

385 390 395 400

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

405 410 415

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

420 425 430

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

435 440 445

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

450 455 460

Gly Gly Ser Gly Tyr Ala Leu His Asp Asp Tyr Tyr Gly Leu Asp Val

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

595 600 605

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

610 615 620

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

625 630 635 640

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

645 650 655

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

660 665 670

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

675 680 685

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

690 695 700

Ala Leu His Met Gln Ala Leu Pro Pro Arg

705 710

<210> 347

<211> 708

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 347

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln

115 120 125

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

130 135 140

Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp

145 150 155 160

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala

165 170 175

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

180 185 190

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

195 200 205

Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly

210 215 220

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

245 250 255

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

260 265 270

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp

275 280 285

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser

290 295 300

Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

305 310 315 320

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

325 330 335

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Glu

340 345 350

Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln

370 375 380

Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser

385 390 395 400

Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln

405 410 415

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu

420 425 430

His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

435 440 445

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr

450 455 460

Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Cys Gly Thr

465 470 475 480

Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

485 490 495

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

500 505 510

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

515 520 525

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

530 535 540

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

545 550 555 560

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

565 570 575

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

580 585 590

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

595 600 605

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

610 615 620

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

625 630 635 640

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

645 650 655

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

660 665 670

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

675 680 685

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

690 695 700

Leu Pro Pro Arg

705

<210> 348

<211> 708

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 348

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Glu Trp Trp Gly Glu Ser Trp Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile

115 120 125

Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

130 135 140

Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

145 150 155 160

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His

165 170 175

Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

180 185 190

Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

195 200 205

Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe

210 215 220

Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

245 250 255

Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser

260 265 270

Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

275 280 285

Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile

290 295 300

Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val

305 310 315 320

Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser

325 330 335

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr

340 345 350

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

370 375 380

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

385 390 395 400

Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln

405 410 415

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu

420 425 430

Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

435 440 445

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

450 455 460

Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Cys Gly Thr

465 470 475 480

Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

485 490 495

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

500 505 510

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

515 520 525

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

530 535 540

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

545 550 555 560

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

565 570 575

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

580 585 590

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

595 600 605

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

610 615 620

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

625 630 635 640

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

645 650 655

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

660 665 670

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

675 680 685

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

690 695 700

Leu Pro Pro Arg

705

<210> 349

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 349

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 350

<211> 465

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 350

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

245 250 255

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

260 265 270

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

275 280 285

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

290 295 300

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

305 310 315 320

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

325 330 335

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

340 345 350

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 351

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 351

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 352

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 352

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacaccagc attcctcctg 60

atccca 66

<210> 353

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 353

atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60

atcccc 66

<210> 354

<211> 1461

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 354

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca 120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag 180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct 240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag 300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga 360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt 420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact 480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc 540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact 600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag 660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag 720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc 780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc 840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc 900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg 960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg 1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt 1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc 1140

agcgcagatg ctccagccta ccagcagggg cagaaccagc tctacaacga actcaatctt 1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag 1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac 1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg 1440

caggccctgc cgcctcggta a 1461

<210> 355

<211> 1458

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 355

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca 120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag 180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct 240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag 300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga 360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt 420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact 480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc 540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact 600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag 660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag 720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc 780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc 840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc 900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg 960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg 1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt 1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc 1140

agcgcagatg ctccagccta ccagcagggg cagaaccagc tctacaacga actcaatctt 1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc 1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag 1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac 1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg 1440

caggccctgc cgcctcgg 1458

<210> 356

<211> 1398

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 356

gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60

ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120

ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180

aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240

gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300

ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360

ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420

tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480

cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540

taccaatcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600

tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660

tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720

tccagcacca ctaccccagc accgaggcca cccaccccgg ctcctaccat cgcctcccag 780

cctctgtccc tgcgtccgga ggcatgtaga cccgcagctg gtggggccgt gcatacccgg 840

ggtcttgact tcgcctgcga tatctacatt tgggcccctc tggctggtac ttgcggggtc 900

ctgctgcttt cactcgtgat cactctttac tgtaagcgcg gtcggaagaa gctgctgtac 960

atctttaagc aacccttcat gaggcctgtg cagactactc aagaggagga cggctgttca 1020

tgccggttcc cagaggagga ggaaggcggc tgcgaactgc gcgtgaaatt cagccgcagc 1080

gcagatgctc cagcctacca gcaggggcag aaccagctct acaacgaact caatcttggt 1140

cggagagagg agtacgacgt gctggacaag cggagaggac gggacccaga aatgggcggg 1200

aagccgcgca gaaagaatcc ccaagagggc ctgtacaacg agctccaaaa ggataagatg 1260

gcagaagcct atagcgagat tggtatgaaa ggggaacgca gaagaggcaa aggccacgac 1320

ggactgtacc agggactcag caccgccacc aaggacacct atgacgctct tcacatgcag 1380

gccctgccgc ctcggtaa 1398

<210> 357

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 357

caggtccagc tgcaggaatc aggaccaggg ctggtgaaac ctagcgaaac tctgagtctg 60

acttgtaccg tctccggggt gtctctgcca gactacggcg tgagctggat cagacagccc 120

cctggcaagt gcctggagtg gatcggcgtg atctggggct ccgagaccac atactatcag 180

agctccctga agtctcgggt gaccatctcc aaggacaact ctaagaatca ggtgagcctg 240

aagctgtcta gcgtgaccgc cgccgataca gccgtgtact attgtgccaa gcactactat 300

tacggcggct cctatgccat ggattactgg ggccagggca ccctggtgac agtgtcctct 360

<210> 358

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 358

caggtccagc tgcaggaatc cggcccagga ctggtgaagc ctagcgagac cctgtccctg 60

acctgcacag tgagcggcgt gtccctgccc gattacggcg tgagctggat cagacagccc 120

cctggcaagt gtctggagtg gatcggcgtg atctggggct ctgagaccac atactatcag 180

tcctctctga agagcagggt gaccatctct aaggacaaca gcaagaatca ggtgtccctg 240

aagctgagct ccgtgaccgc agcagataca gccgtgtact attgcgccaa gcactactat 300

tacggcggct cctatgctat ggattattgg gggcagggca ctctggtcac tgtctcatca 360

<210> 359

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 359

caggtgcagc tgcaggaatc tggacccgga ctggtgaaac ctagtgaaac tctgtctctg 60

acttgtaccg tctcaggggt ctcactgcca gactacggcg tgtcctggat cagacagccc 120

cctggcaagt gcctggagtg gatcggcgtg atctggggct ctgagaccac atactatcag 180

agctccctga agagccgggt gaccatctcc aaggacaact ctaagaatca ggtgtccctg 240

aagctgtcta gcgtgaccgc cgccgataca gccgtgtact attgtgccaa gcactactat 300

tacggcggca gctatgccat ggattactgg ggccagggca ccctggtgac agtgtcctct 360

<210> 360

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 360

caggtccagc tgcaggaaag cggcccagga ctggtgaagc ctagcgagac cctgtccctg 60

acctgcacag tgagcggcgt gtccctgcct gattacggcg tgtcctggat cagacagccc 120

cctggcaagt gtctggagtg gatcggcgtg atctggggct ccgagaccac atactatcag 180

tcctctctga agtctagggt gacaatctct aaggacaaca gcaagaatca ggtgagcctg 240

aagctgagct ccgtgaccgc agcagataca gccgtgtact attgtgccaa gcactactat 300

tacggcggct cttatgctat ggattattgg gggcagggca ctctggtcac tgtctcaagc 360

<210> 361

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 361

caggtgcagc tgcaggagag cggcccagga ctggtgaagc cttccgagac actgtctctg 60

acctgtacag tgagcggcgt gtccctgccc gactacggcg tgtcctggat cagacagcca 120

cctggcaagg gactggagtg gatcggcgtg atctggggca gcgagaccac atactatcag 180

agctccctga agtccagggt gaccatcagc aaggacaact ccaagaatca ggtgagcctg 240

aagctgtcta gcgtgaccgc cgccgataca gccgtgtact attgcgccaa gcactactat 300

tacggcggct cctatgccat ggattactgg ggccagggca ccctggtcac agtgtcctct 360

<210> 362

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 362

caggtgcagc tgcaggagtc tggcccagga ctggtgaagc cttctgagac cctgagcctg 60

acctgcacag tgtccggcgt gtctctgccc gattacggcg tgtcctggat cagacagcca 120

cctggcaagg gactggagtg gatcggcgtg atctggggct ctgagaccac atactatcag 180

tctagcctga agagccgggt gacaatctcc aaggacaact ctaagaatca ggtgtccctg 240

aagctgtcct ctgtgaccgc cgccgataca gccgtgtact attgtgccaa gcactactat 300

tacggcggca gctatgccat ggactactgg ggccagggca ccctggtgac agtgagctcc 360

<210> 363

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 363

caggtgcagc tgcaggagtc tggcccagga ctggtgaagc cttctgagac cctgagcctg 60

acctgcacag tgagcggcgt gtccctgccc gattacggcg tgtcctggat cagacagcca 120

cctggcaagg gactggagtg gatcggcgtg atctggggca gcgagaccac atactatcag 180

tcctctctga agtccagggt gacaatctcc aaggacaact ctaagaatca ggtgagcctg 240

aagctgagct ccgtgaccgc agcagataca gccgtgtact attgcgccaa gcactactat 300

tacggcggct cctatgccat ggactactgg ggccagggca ccctggtcac agtgtctagc 360

<210> 364

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 364

caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttccgagac actgtctctg 60

acctgtacag tgtccggcgt gtctctgccc gactacggcg tgagctggat cagacagcca 120

cctggcaagg gactggagtg gatcggcgtg atctggggct ctgagaccac atactatcag 180

tcctctctga agagccgggt gaccatcagc aaggacaact ccaagaatca ggtgtccctg 240

aagctgagct ccgtgaccgc agcagataca gccgtgtact attgcgccaa gcactactat 300

tacggcggca gctatgccat ggattactgg ggccagggca ccctggtgac agtgtctagc 360

<210> 365

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 365

gagatcgtga tgacccagag cccagccaca ctgagcctgt ccccaggaga gagggccaca 60

ctgtcttgta gagccagcca ggatatctcc aagtatctga actggtacca gcagaagcct 120

ggacaggcac caaggctgct gatctaccac acctctagac tgcacagcgg catccctgcc 180

aggttttctg gcagcggctc cggcacagac tataccctga caatctctag cctgcagcca 240

gaggatttcg ccgtgtactt ttgtcagcag ggcaatactc tgccatacac ctttggatgc 300

ggaactaaac tggaaatcaa g 321

<210> 366

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 366

gaaattgtga tgacccagtc ccccgctact ctgtctctgt cccccggaga acgggctact 60

ctgtcttgtc gcgcttccca ggatattagc aagtacctga actggtatca gcagaagcca 120

ggacaggcac caaggctgct gatctaccac acctctcgcc tgcacagcgg catccctgca 180

cggttctctg gcagcggctc cggcacagac tacaccctga caatcagctc cctgcagcct 240

gaggatttcg ccgtgtactt ttgccagcag ggcaataccc tgccatatac atttggctgt 300

ggcaccaagc tggagatcaa g 321

<210> 367

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 367

gagatcgtga tgacccagtc cccagccaca ctgagcctgt ccccaggaga gagggccacc 60

ctgtcttgta gagccagcca ggatatctcc aagtatctga actggtacca gcagaagcct 120

ggacaggcac caaggctgct gatctaccac acctctagac tgcacagcgg catccctgcc 180

aggttttctg gcagcggctc cggcacagac tataccctga caatctctag cctgcagcca 240

gaggatttcg ccgtgtactt ttgtcagcag ggaaatactc tgccatacac ctttggatgc 300

ggaactaaac tggaaatcaa g 321

<210> 368

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 368

gagattgtga tgacccagtc ccccgccacc ctgagtctga gccccggaga acgagctacc 60

ctgagttgcc gagcttccca ggacatttcc aagtacctga actggtatca gcagaagcca 120

ggacaggcac caaggctgct gatctaccac acctctcgcc tgcacagcgg catcccagca 180

cggttctctg gcagcggctc cggcacagac tacaccctga caatcagctc cctgcagcct 240

gaggatttcg ccgtgtactt ttgccagcag ggcaataccc tgccatatac atttggctgt 300

ggcaccaagc tggagatcaa g 321

<210> 369

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 369

gagatcgtga tgacccagtc tccagccaca ctgtctctga gcccaggaga gagggccacc 60

ctgtcttgcc gcgccagcca ggatatctcc aagtatctga actggtacca gcagaagcca 120

ggacaggcac caaggctgct gatctaccac acctctcgcc tgcacagcgg catcccagca 180

cggttctccg gctctggcag cggcacagac tacaccctga caatctcctc tctgcagccc 240

gaggatttcg ccgtgtattt ttgccagcag ggcaataccc tgccttacac atttggccag 300

ggcaccaagc tggagatcaa g 321

<210> 370

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 370

gagatcgtga tgacccagag cccagccaca ctgagcctgt ccccaggaga gagggccacc 60

ctgagctgca gagcctccca ggatatctct aagtatctga actggtacca gcagaagcct 120

ggacaggcac caaggctgct gatctaccac accagcagac tgcactccgg catccctgca 180

aggttctctg gcagcggctc cggcacagac tacaccctga caatctctag cctgcagcct 240

gaggatttcg ccgtgtattt ttgtcagcag ggcaataccc tgccatacac atttggccag 300

ggcaccaagc tggagatcaa g 321

<210> 371

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 371

gagatcgtga tgacccagag cccagccaca ctgtctctga gcccaggaga gagggccacc 60

ctgagctgtc gcgcctccca ggatatctct aagtatctga actggtacca gcagaagcca 120

ggacaggcac caaggctgct gatctaccac accagccgcc tgcactccgg catcccagca 180

cggttctccg gctctggcag cggcacagac tacaccctga caatctcctc tctgcagccc 240

gaggatttcg ccgtgtattt ttgccagcag ggcaataccc tgccttacac atttggccag 300

ggcaccaagc tggagatcaa g 321

<210> 372

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 372

gagatcgtga tgacccagtc tccagccaca ctgagcctgt ccccaggaga gagggccacc 60

ctgtcttgca gagccagcca ggatatctcc aagtatctga actggtacca gcagaagcct 120

ggacaggcac caaggctgct gatctaccac acctctagac tgcacagcgg catcccagca 180

aggttctctg gcagcggctc cggcacagac tacaccctga caatctctag cctgcagcct 240

gaggatttcg ccgtgtattt ttgccagcag ggcaataccc tgccatacac atttggccag 300

ggcaccaagc tggagatcaa g 321

<210> 373

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 373

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 374

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 374

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 375

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 375

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 376

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 376

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 377

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 377

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 378

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 378

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 379

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 379

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 380

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 380

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 381

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 381

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 382

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 382

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 383

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 383

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 384

<211> 1467

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 384

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacaccagc attcctcctg 60

atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120

gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180

aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240

ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300

gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360

ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420

ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480

ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540

gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600

agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660

tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720

tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780

acctcagtca ccgtctcctc agcggccgca attgaagtta tgtatcctcc tccttaccta 840

gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 900

cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg 960

gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac cagcaggggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgc 1467

<210> 385

<211> 489

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 385

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Ile Glu

260 265 270

Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr

275 280 285

Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro

290 295 300

Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu

305 310 315 320

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

325 330 335

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

340 345 350

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

355 360 365

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 386

<211> 801

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 386

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacaccagc attcctcctg 60

atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120

gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180

aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240

ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300

gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360

ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct 420

ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg 480

ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt 540

gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt 600

agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac 660

tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac 720

tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga 780

acctcagtca ccgtctcctc a 801

<210> 387

<211> 266

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 387

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser

260 265

<210> 388

<211> 1401

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 388

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag 360

ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 420

cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 480

tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 540

accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 600

agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 660

gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 720

gtcaccgtct cctcagcggc cgcaattgaa gttatgtatc ctcctcctta cctagacaat 780

gagaagagca atggaaccat tatccatgtg aaagggaaac acctttgtcc aagtccccta 840

tttcccggac cttctaagcc cttttgggtg ctggtggtgg ttgggggagt cctggcttgc 900

tatagcttgc tagtaacagt ggcctttatt attttctggg tgaggagtaa gaggagcagg 960

ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1020

taccagccct atgccccacc acgcgacttc gcagcctatc gctccagagt gaagttcagc 1080

aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1140

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1200

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1260

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1320

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1380

atgcaggccc tgccccctcg c 1401

<210> 389

<211> 467

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 389

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro

245 250 255

Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly

260 265 270

Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe

275 280 285

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

290 295 300

Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg

305 310 315 320

Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro

325 330 335

Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala

340 345 350

Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

355 360 365

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

370 375 380

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

385 390 395 400

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

405 410 415

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

420 425 430

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

435 440 445

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

450 455 460

Pro Pro Arg

465

<210> 390

<211> 735

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 390

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag 360

ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 420

cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 480

tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 540

accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 600

agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 660

gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 720

gtcaccgtct cctca 735

<210> 391

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 391

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 392

<211> 1383

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 392

atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60

atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120

gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180

aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240

cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300

gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360

ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420

ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480

cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540

gtgagctgga tccggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600

agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660

agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720

tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780

accagcgtga ccgtgagcag cgaatctaag tacggaccgc cctgcccccc ttgccctatg 840

ttctgggtgc tggtggtggt cggaggcgtg ctggcctgct acagcctgct ggtcaccgtg 900

gccttcatca tcttttgggt gaaacggggc agaaagaaac tcctgtatat attcaaacaa 960

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1020

gaagaagaag aaggaggatg tgaactgcgg gtgaagttca gcagaagcgc cgacgcccct 1080

gcctaccagc agggccagaa tcagctgtac aacgagctga acctgggcag aagggaagag 1140

tacgacgtcc tggataagcg gagaggccgg gaccctgaga tgggcggcaa gcctcggcgg 1200

aagaaccccc aggaaggcct gtataacgaa ctgcagaaag acaagatggc cgaggcctac 1260

agcgagatcg gcatgaaggg cgagcggagg cggggcaagg gccacgacgg cctgtatcag 1320

ggcctgtcca ccgccaccaa ggatacctac gacgccctgc acatgcaggc cctgccccca 1380

agg 1383

<210> 393

<211> 461

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 393

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly

260 265 270

Pro Pro Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly

275 280 285

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

290 295 300

Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

305 310 315 320

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

325 330 335

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

340 345 350

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

355 360 365

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

370 375 380

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

385 390 395 400

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

405 410 415

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

420 425 430

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

435 440 445

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

450 455 460

<210> 394

<211> 801

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 394

atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ctttctgctg 60

atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct gggcgaccgg 120

gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg gtatcagcag 180

aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca cagcggcgtg 240

cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat ctccaacctg 300

gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc ctacaccttt 360

ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa gcctggcagc 420

ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg cctggtggcc 480

cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc cgactacggc 540

gtgagctgga tccggcagcc ccccaggaag ggcctggaat ggctgggcgt gatctggggc 600

agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat caaggacaac 660

agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac cgccatctac 720

tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg gggccagggc 780

accagcgtga ccgtgagcag c 801

<210> 395

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 395

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

260 265

<210> 396

<211> 1317

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 396

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagcgaatc taagtacgga ccgccctgcc ccccttgccc tatgttctgg 780

gtgctggtgg tggtcggagg cgtgctggcc tgctacagcc tgctggtcac cgtggccttc 840

atcatctttt gggtgaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 900

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 960

gaagaaggag gatgtgaact gcgggtgaag ttcagcagaa gcgccgacgc ccctgcctac 1020

cagcaggggcc agaatcagct gtacaacgag ctgaacctgg gcagaaggga agagtacgac 1080

gtcctggata agcggagagg ccgggaccct gagatgggcg gcaagcctcg gcggaagaac 1140

ccccaggaag gcctgtataa cgaactgcag aaagacaaga tggccgaggc ctacagcgag 1200

atcggcatga agggcgagcg gaggcggggc aagggccacg acggcctgta tcagggcctg 1260

tccaccgcca ccaaggatac ctacgacgcc ctgcacatgc aggccctgcc cccaagg 1317

<210> 397

<211> 439

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 397

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

245 250 255

Pro Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr

260 265 270

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly

275 280 285

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

290 295 300

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

305 310 315 320

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

325 330 335

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

340 345 350

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

355 360 365

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

370 375 380

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

385 390 395 400

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

405 410 415

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

420 425 430

Met Gln Ala Leu Pro Pro Arg

435

<210> 398

<211> 735

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 398

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagc 735

<210> 399

<211> 245

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 399

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 400

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 400

caggtgcagc tgcaggagtc cggcggcggc gtggtgcagc caggccggtc cctgagactg 60

tcttgtgccg ccagcggctt caccttttcc tcttatggca tgcactgggt gagacaggca 120

cctggcaagg gactggagtg ggtggccgtg atctcctacg acggctctaa caagtattac 180

gccgatagcg tgaagggcag gttcaccatc agccgcgaca actccaagaa tacactgtat 240

ctgcagatga atagcctgcg ggccgaggat accgccgtgt attactgcgg aggctccggc 300

tacgcactgc acgacgatta ttacggactg gacgtgtggg gacagggcac cctggtcaca 360

gtgagctcc 369

<210> 401

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 401

caggtgcagc tgcaggagtc tggcggagga gtggtgcagc caggccggtc cctgagactg 60

tcttgcgccg ccagcggctt cacattttct agctacggaa tgcactgggt gcgccaggca 120

cctggcaagg gactggagtg ggtggccgtg atctcctatg acggctctaa caagtactat 180

gccgattccg tgaagggcag gttcaccatc agccgcgaca actccaagaa tacactgtac 240

ctgcagatga attccctgcg ggccgaggat accgccgtgt actattgtgg cggctctggc 300

tatgccctgc acgacgatta ctatggactg gacgtgtggg gacagggcac cctggtgaca 360

gtgtcctct 369

<210> 402

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 402

caggtgcagc tgcaggagtc tggcggagga gtggtgcagc caggccggag cctgagactg 60

tcctgcgccg cctctggctt cacctttagc tcctatggca tgcactgggt gagacaggca 120

cctggcaagg gactggagtg ggtggccgtg atcagctacg acggctccaa caagtattac 180

gccgatagcg tgaagggcag gttcaccatc tctcgcgaca acagcaagaa tacactgtat 240

ctgcagatga attccctgcg ggccgaggat acagccgtgt attactgcgg aggcagcggc 300

tacgcactgc acgacgatta ttacggactg gacgtgtggg gacagggcac cctggtcaca 360

gtgtctagc 369

<210> 403

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 403

caggtgcagc tgcaggagag cggcggcggc gtggtgcagc ccggccggtc tctgagactg 60

agctgtgccg cctccggctt cacctttagc tcctacggaa tgcactgggt gcgccaggca 120

cctggcaagg gactggagtg ggtggccgtg atctcttatg acggcagcaa caagtactat 180

gccgatagcg tgaagggcag gttcaccatc tcccgcgaca actctaagaa tacactgtac 240

ctgcagatga atagcctgcg ggccgaggat accgccgtgt actattgcgg aggctccggc 300

tatgcactgc acgacgatta ctatggactg gacgtgtggg gacagggcac cctggtgaca 360

gtgtctagc 369

<210> 404

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 404

caggtccagc tgcaggagag tggggggggg gtcgtccagc ccggaagaag cctgagactg 60

tcatgtgccg catctgggtt tacttttagc tcctatggaa tgcactgggt gcgccaggca 120

cctggcaagt gcctggagtg ggtggccgtg atctcctacg acggctctaa caagtactat 180

gccgatagcg tgaagggccg gttcaccatc agcagagaca actccaagaa tacactgtat 240

ctgcagatga attctctgcg ggccgaggat accgccgtgt actattgtgg aggctccggc 300

tacgcactgc acgacgatta ctatggactg gacgtgtggg gacagggcac cctggtgaca 360

gtgtctagc 369

<210> 405

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 405

caggtccagc tgcaggaatc cggcggagga gtggtgcagc caggccggtc tctgagactg 60

agctgcgccg cctccggctt cacattttcc tcttatggca tgcactgggt gagacaggcc 120

cctggcaagt gtctggagtg ggtggccgtg atctcttacg acggcagcaa caagtattac 180

gccgatagcg tgaagggcag gttcaccatc tcccgcgaca actctaagaa tacactgtat 240

ctgcagatga attccctgcg ggccgaggat accgccgtgt attactgcgg cggctctggc 300

tacgccctgc acgacgacta ctatggactg gatgtctggg ggcagggcac actggtcact 360

gtctcttca 369

<210> 406

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 406

caggtccagc tgcaggaatc aggggggggg gtcgtccagc ccggaagaag tctgagactg 60

tcatgtgccg catcagggtt tacctttagc tcctatggaa tgcactgggt gcgccaggca 120

cctggcaagt gcctggagtg ggtggccgtg atctcctacg acggctctaa caagtactat 180

gccgatagcg tgaagggccg gttcaccatc agcagagaca actccaagaa tacactgtat 240

ctgcagatga attctctgcg ggccgaggat accgccgtgt actattgtgg aggctccggc 300

tacgcactgc acgacgatta ctatggactg gacgtgtggg gacagggcac cctggtgaca 360

gtgtctagc 369

<210> 407

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 407

caggtccagc tgcaggaatc cggcggagga gtggtgcagc caggccggtc tctgagactg 60

agctgcgccg cctccggctt caccttttcc tcttatggca tgcactgggt gagacaggcc 120

cctggcaagt gtctggagtg ggtggccgtg atctcttacg acggcagcaa caagtattac 180

gccgatagcg tgaagggcag gttcaccatc tcccgcgaca actctaagaa tacactgtat 240

ctgcagatga attccctgcg ggccgaggat acagccgtgt attactgtgg cggctctggc 300

tacgccctgc atgatgatta ttatggactg gatgtctggg ggcagggcac actggtcact 360

gtctcttcc 369

<210> 408

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 408

cagtctgccc tgacccagcc agcaagcgtg tccggctctc ctggccagag catcacaatc 60

tcctgcaccg gcacaagctc cgacgtggga ggctataact acgtgagctg gtatcagcag 120

cacccaggca aggcccccaa gctgatgatc tacgacgtga gcaacaggcc ttctggcgtg 180

agcaatcgct tcagcggctc caagtctggc aataccgcct ctctgacaat cagcggcctg 240

caggcagagg acgaggcaga ttattactgc tctagctata cctcctctag cacactgtac 300

gtgtttggca gcggcaccaa ggtgacagtg ctg 333

<210> 409

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 409

cagagcgccc tgacccagcc agcatccgtg tctggcagcc caggccagtc tatcacaatc 60

agctgcaccg gcacaagctc cgacgtggga ggctacaact atgtgagctg gtaccagcag 120

caccctggca aggccccaaa gctgatgatc tatgacgtga gcaaccggcc atccggcgtg 180

tctaatagat tctccggctc taagagcggc aataccgcct ccctgacaat ctctggcctg 240

caggcagagg acgaggcaga ttactattgt tctagctaca cctcctctag cacactgtac 300

gtgttcggca gcggcaccaa ggtgacagtg ctg 333

<210> 410

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 410

cagtctgccc tgacccagcc agcaagcgtg tccggctctc ctggccagtc catcacaatc 60

tcttgtaccg gcacatcctc tgacgtgggc ggctataact acgtgtcctg gtatcagcag 120

cacccaggca aggcccccaa gctgatgatc tacgatgtga gcaacaggcc ttctggcgtg 180

agcaatcgct tcagcggctc caagtctggc aataccgcca gcctgacaat ctccggcctg 240

caggcagagg acgaggcaga ttattactgc agctcctata cctctagctc cacactgtac 300

gtgtttggca gcggcaccaa ggtgacagtg ctg 333

<210> 411

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 411

cagagcgccc tgacccagcc agcatccgtg tctggcagcc caggccagtc catcacaatc 60

tcttgcaccg gcacatctag cgacgtgggc ggctacaact acgtgagctg gtaccagcag 120

caccctggca aggccccaaa gctgatgatc tatgatgtga gcaaccggcc ctccggcgtg 180

tctaatagat tctccggctc taagagcggc aataccgcca gcctgacaat ctccggcctg 240

caggcagagg acgaggcaga ttactattgc tcctcttaca ccagctcctc tacactgtac 300

gtgttcggct ccggcaccaa ggtgacagtg ctg 333

<210> 412

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 412

cagtctgccc tgacccagcc tgcaagcgtg tccggctctc caggccagtc tatcacaatc 60

agctgtaccg gcacaagctc cgacgtgggc ggctataact acgtgagctg gtatcagcag 120

caccctggca aggccccaaa gctgatgatc tacgacgtga gcaaccggcc ctctggcgtg 180

agcaatcggt tcagcggcag caagtctggc aataccgcct ccctgacaat ctctggcctg 240

caggcagagg acgaggcaga ttattactgt agcagttata cttcaagctc aaccctgtac 300

gtgtttggat gcggcactaa ggtcaccgtc ctg 333

<210> 413

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 413

cagtctgctc tgacccagcc cgcttccgtc tcagggtctc caggacagtc aattaccatt 60

agttgcacag gcacctcatc cgatgtgggc ggctataact acgtgtcctg gtatcagcag 120

cacccaggca aggcccccaa gctgatgatc tacgacgtga gcaacaggcc atctggcgtg 180

agcaatcgct tcagcggctc caagtctggc aataccgcca gcctgacaat ctccggcctg 240

caggcagagg acgaggcaga ttactattgc agctcctata cctctagctc cacactgtac 300

gtgtttggct gtggcaccaa ggtgacagtg ctg 333

<210> 414

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 414

cagtctgccc tgacccagcc tgcaagcgtg tccggctctc caggccagtc tatcacaatc 60

agctgtaccg gcacaagctc cgacgtgggc ggctataact acgtgagctg gtatcagcag 120

caccctggca aggccccaaa gctgatgatc tacgacgtga gcaaccggcc ctctggcgtg 180

agcaatcggt tcagcggcag caagtctggc aataccgcct ccctgacaat ctctggcctg 240

caggcagagg acgaggcaga ttattactgt agctcctaca cttcttcaag cacactgtat 300

gtctttggat gcggaactaa ggtcactgtc ctg 333

<210> 415

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 415

cagtctgctc tgacccagcc cgcttccgtc tcaggatctc caggacagtc tattacaatt 60

agttgcacag gaacctcttc cgatgtgggc ggctataact acgtgtcctg gtatcagcag 120

cacccaggca aggcccccaa gctgatgatc tacgacgtga gcaacaggcc ttctggcgtg 180

agcaatcgct tcagcggctc caagtctggc aataccgcca gcctgacaat ctccggcctg 240

caggcagagg acgaggcaga ttactattgc agctcctata cctctagctc cacactgtac 300

gtgtttggct gtggcaccaa ggtgacagtg ctg 333

<210> 416

<211> 1485

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 416

atggccctcc ctgtcaccgc tctgttgctg ccgcttgctc tgctgctcca cgcagcgcga 60

ccgcaggtac aattgcagga gtctggaggc ggtgtggtgc aacccggtcg cagcttgcgc 120

ctgagttgtg ctgcgtctgg atttacattt tcatcttacg gaatgcattg ggtacgccag 180

gcaccgggga aaggccttga atgggtggct gtaatttcat acgatggttc caacaaatac 240

tatgctgact cagtcaaggg tcgatttaca attagtcggg acaactccaa gaacaccctt 300

tatcttcaaa tgaattccct tagagcagag gatacggcgg tctattactg tggtggcagt 360

ggttatgcac ttcatgatga ttactatggc ttggatgtct gggggcaagg gacgcttgta 420

actgtatcct ctggtggtgg tggtagtggt gggggaggct ccggcggtgg cggctctcaa 480

tctgctctga ctcaaccagc aagcgtatca gggtcaccgg gacagagtat taccataagt 540

tgcacgggga cctctagcga tgtagggggg tataattatg tatcttggta tcaacaacac 600

cccgggaaag cccctaaatt gatgatctac gacgtgagca atcgacctag tggcgtatca 660

aatcgcttct ctggtagcaa gagtgggaat acggcgtccc ttactattag cggattgcaa 720

gcagaagatg aggccgatta ctactgcagc tcctatacta gctcttctac attgtacgtc 780

tttgggagcg gaacaaaagt aacagtactc acaacaacac ctgccccgag accgcctaca 840

ccagccccga ctattgccag ccagcctctg agcctcaggc ctgaggcctg taggcccgca 900

gcgggcggcg cagttcatac acggggcttg gatttcgctt gtgatattta tatttgggct 960

cctttggcgg ggacatgtgg cgtgctgctt ctgtcacttg ttattacact gtactgtaaa 1020

cgcgggcgaa aaaaattgct gtatattttt aagcagccat ttatgaggcc cgttcagacg 1080

acgcaggagg aggacggttg ctcttgcagg ttcccagaag aggaagaagg gggctgtgaa 1140

ttgcgggtta aattttcaag atccgcagac gctccagcat accaacaggg acaaaaccaa 1200

ctctataacg agctgaatct tggaagaagg gaggaatatg atgtgctgga taaacggcgc 1260

ggtagagatc cggagatggg cggaaaacca aggcgaaaaa accctcagga gggactctac 1320

aacgaactgc agaaagacaa aatggcggag gcttattccg aaataggcat gaagggcgag 1380

cggaggcgag ggaaagggca cgacggactg tatcaaggcc tctcaaccgc gactaaggat 1440

acgtacgacg ccctgcacat gcaggccctg cctccgagat gataa 1485

<210> 417

<211> 1395

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 417

gaaattgtga tgacccagtc acccgccact cttagccttt cacccggtga gcgcgcaacc 60

ctgtcttgca gagcctccca agacatctca aaatacctta attggtatca acagaagccc 120

ggacaggctc ctcgccttct gatctaccac accagccggc tccattctgg aatccctgcc 180

aggttcagcg gtagcggatc tgggaccgac tacaccctca ctatcagctc actgcagcca 240

gaggacttcg ctgtctattt ctgtcagcaa gggaacaccc tgccctacac ctttggacag 300

ggcaccaagc tcgagattaa aggtggaggt ggcagcggag gaggtgggtc cggcggtgga 360

ggaagccagg tccaactcca agaaagcgga ccgggtcttg tgaagccatc agaaactctt 420

tcactgactt gtactgtgag cggagtgtct ctccccgatt acggggtgtc ttggatcaga 480

cagccaccgg ggaagggtct ggaatggatt ggagtgattt ggggctctga gactacttac 540

taccaatcat ccctcaagtc acgcgtcacc atctcaaagg acaactctaa gaatcaggtg 600

tcactgaaac tgtcatctgt gaccgcagcc gacaccgccg tgtactattg cgctaagcat 660

tactattatg gcgggagcta cgcaatggat tactggggac agggtactct ggtcaccgtg 720

tccagcacca ctaccccagc accgaggcca cccaccccgg ctcctaccat cgcctcccag 780

cctctgtccc tgcgtccgga ggcatgtaga cccgcagctg gtggggccgt gcatacccgg 840

ggtcttgact tcgcctgcga tatctacatt tgggcccctc tggctggtac ttgcggggtc 900

ctgctgcttt cactcgtgat cactctttac tgtaagcgcg gtcggaagaa gctgctgtac 960

atctttaagc aacccttcat gaggcctgtg cagactactc aagaggagga cggctgttca 1020

tgccggttcc cagaggagga ggaaggcggc tgcgaactgc gcgtgaaatt cagccgcagc 1080

gcagatgctc cagcctacca gcaggggcag aaccagctct acaacgaact caatcttggt 1140

cggagagagg agtacgacgt gctggacaag cggagaggac gggacccaga aatgggcggg 1200

aagccgcgca gaaagaatcc ccaagagggc ctgtacaacg agctccaaaa ggataagatg 1260

gcagaagcct atagcgagat tggtatgaaa ggggaacgca gaagaggcaa aggccacgac 1320

ggactgtacc agggactcag caccgccacc aaggacacct atgacgctct tcacatgcag 1380

gccctgccgc ctcgg 1395

<---

Claims (143)

1. A T cell or NK cell for expressing a chimeric antigen receptor (CAR) molecule, wherein the cell comprises a CAR molecule, wherein said CAR molecule comprises: (a) a first CAR comprising a first antigen-binding domain that binds to BCMA (BCMA CAR), a first transmembrane domain, and a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19 (CD19 CAR), a second transmembrane domain, and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Where: (i) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 44, 45, 76, 54, 55 and 56, respectively and the second CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (ii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 47, 48, 76, 57, 58 and 59, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 310, 311, 246, 316, 317 and 318, respectively; (iii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 86, 87, 88, 95, 96 and 97, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (iv) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 47, 89, 88, 98, 99 and 100, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (v) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 44, 45, 46, 54, 55 and 56, respectively and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (vi) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 47, 48, 46, 57, 58 and 59, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (vii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 231, 232, 233, 54, 55 and 240, respectively and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (viii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 234, 235, 233, 57, 58 and 241, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (ix) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC of SEQ ID NOs: 295, 245, 246, 247, 248, and 249, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC, and LCDR3 LC as set forth in one of SEQ ID NOs: 231, 232, 233, 54, 55, and 240, respectively; or (x) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC of SEQ ID NOs: 310, 311, 246, 316, 317, and 318, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC, and LCDR3 LC as set forth in one of SEQ ID NOs: 234, 235, 233, 57, 58, and 241, respectively; and wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, 222 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto. 2. The cell of claim 1, wherein the first CAR is encoded by a first nucleic acid sequence and the second CAR is encoded by a second nucleic acid sequence, wherein the first and second nucleic acid sequences are located on the same nucleic acid molecule. 3. The cell according to item 2, in which: (a) One nucleic acid molecule has the following configuration in 5'-3' orientation: (i) a nucleic acid sequence encoding a first antigen-binding domain, - a nucleic acid sequence encoding a first transmembrane domain, - a nucleic acid sequence encoding a first intracellular signaling domain, - a nucleic acid sequence encoding a linker, - a nucleic acid sequence encoding a second antigen-binding domain, - a nucleic acid sequence encoding a second transmembrane domain, - a nucleic acid sequence encoding a second intracellular signaling domain; or (ii) a nucleic acid sequence encoding a second antigen-binding domain, - a nucleic acid sequence encoding a second transmembrane domain, - a nucleic acid sequence encoding a second intracellular signaling domain, - a nucleic acid sequence encoding a linker, - a nucleic acid sequence encoding a first antigen-binding domain, - a nucleic acid sequence encoding a first transmembrane domain, - a nucleic acid sequence encoding a first intracellular signaling domain, wherein the linker optionally comprises a self-cleavage site, wherein the linker optionally comprises a P2A site, a T2A site, an E2A site, or an F2A site, wherein the linker optionally comprises a P2A site, optionally wherein: (I) the linker is encoded by a nucleic acid sequence of SEQ ID NO: 209 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, or (II) the linker comprises the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto; (b) one nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 215, 217, 219, 221 or 223 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto; or (c) one nucleic acid molecule encodes an amino acid sequence of SEQ ID NO: 214, 216, 218, 220 or 222 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. 4. The cell of claim 1, wherein the first antigen-binding domain or the second antigen-binding domain comprises VH and VL, wherein the VH and VL are connected by a linker, wherein the linker comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. 5. The cell according to item 2, in which: (i) the first transmembrane domain or the second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto; (ii) the first transmembrane domain or the second transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (iii) the first antigen-binding domain is connected to the first transmembrane domain via a first hinge region and/or the second antigen-binding domain is connected to the second transmembrane domain via a second hinge region, optionally wherein: (a) the hinge region comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (b) the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (c) the hinge region and the transmembrane domain comprise the amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the hinge region and the transmembrane domain are encoded by the nucleic acid sequence of SEQ ID NO: 203 or 213 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (iv) the primary signaling domain contains a functional signaling domain derived from CD3-zeta, where: (a) the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the primary signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 20, 21 or 205, a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto; (v) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB (CD137), where: (a) the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 18 or 204, a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (vi) the first intracellular signaling domain or the second intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3-zeta, wherein: (a) the first intracellular signaling domain or the second intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto and the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the first intracellular signaling domain or the second intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10; or (vii) the first CAR further comprises a first leader sequence or the second CAR further comprises a second leader sequence, where: (a) the first or second leader sequence comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (b) the first or second leader sequence is encoded by the nucleic acid sequence of SEQ ID NO: 199 or 210 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. 6. The cell according to item 5, where: (a) (i) the first leader sequence and the second leader sequence are encoded by different nucleic acid sequences; (ii) the first hinge region and the second hinge region are encoded by different nucleic acid sequences; (iii) the first transmembrane domain and the second transmembrane domain are encoded by different nucleic acid sequences; and/or (iv) the first intracellular signaling domain and the second intracellular signaling domain are encoded by different nucleic acid sequences; (b) (i) the first leader sequence and the second leader sequence comprise the same amino acid sequence or comprise different amino acid sequences; (ii) the first hinge region and the second hinge region comprise the same amino acid sequence or comprise different amino acid sequences; (iii) the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence or comprise different amino acid sequences; and/or (iv) the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequence or comprise different amino acid sequences, optionally where: (I) the first primary signaling domain and the second primary signaling domain are encoded by different nucleic acid sequences; (II) the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by different nucleic acid sequences; (III) the first leader sequence and the second leader sequence comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (IV) the first hinge region and the second hinge region comprise the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto; (V) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto; (VI) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (VII) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (c) (i) the first leader sequence and the second leader sequence are encoded by nucleic acid sequences comprising SEQ ID NOs: 199 and 210, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto or SEQ ID NOs: 210 and 199, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (ii) the first hinge region and the second hinge region are encoded by nucleic acid sequences comprising SEQ ID NOs: 337 and 13, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto or SEQ ID NOs: 13 and 337, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (iii) the first transmembrane domain and the second transmembrane domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 338 and 17, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto or SEQ ID NOs: 17 and 338, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; (iv) the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 204 and 18, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto or SEQ ID NOs: 18 and 204, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; and/or (v) the first primary signaling domain and the second primary signaling domain are encoded by nucleic acid sequences comprising SEQ ID NOs: 205 and 21, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto or SEQ ID NOs: 21 and 205, respectively, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto; or (d) the first CAR or the second CAR is encoded by a nucleic acid molecule that includes the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). 7. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, wherein said nucleic acid molecule comprises: (a) a first nucleic acid sequence encoding a first CAR comprising a first antigen-binding domain that binds to BCMA (BCMA CAR), a first transmembrane domain, and a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second nucleic acid sequence encoding a second CAR comprising a second antigen-binding domain that binds to CD19 (CD19 CAR), a second transmembrane domain, and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Where: (i) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 44, 45, 76, 54, 55 and 56, respectively and the second CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (ii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 47, 48, 76, 57, 58 and 59, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 310, 311, 246, 316, 317 and 318, respectively; (iii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 86, 87, 88, 95, 96 and 97, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (iv) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 47, 89, 88, 98, 99 and 100, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (v) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 44, 45, 46, 54, 55 and 56, respectively and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (vi) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 47, 48, 46, 57, 58 and 59, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (vii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 231, 232, 233, 54, 55 and 240, respectively and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (viii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 234, 235, 233, 57, 58 and 241, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (ix) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC of SEQ ID NOs: 295, 245, 246, 247, 248, and 249, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC, and LCDR3 LC as set forth in one of SEQ ID NOs: 231, 232, 233, 54, 55, and 240, respectively; or (x) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC of SEQ ID NOs: 310, 311, 246, 316, 317, and 318, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC, and LCDR3 LC as set forth in one of SEQ ID NOs: 234, 235, 233, 57, 58, and 241, respectively; and wherein the encoded CAR molecule comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, 222 or an amino acid sequence characterized by at least 95%, 96%, 97%, 98% or 99% identity thereto; and wherein the first nucleic acid sequence and the second nucleic acid sequence are located on a single nucleic acid molecule. 8. An isolated CAR molecule that binds to BCMA and CD19, wherein the CAR molecule comprises: (a) a first CAR comprising a first antigen-binding domain that binds to BCMA (BCMA CAR), a first transmembrane domain, and a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19 (CD19 CAR), a second transmembrane domain, and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Where: (i) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 44, 45, 76, 54, 55 and 56, respectively and the second CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (ii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 47, 48, 76, 57, 58 and 59, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 310, 311, 246, 316, 317 and 318, respectively; (iii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 86, 87, 88, 95, 96 and 97, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (iv) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 47, 89, 88, 98, 99 and 100, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (v) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 44, 45, 46, 54, 55 and 56, respectively and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (vi) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 47, 48, 46, 57, 58 and 59, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (vii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NO: 231, 232, 233, 54, 55 and 240, respectively and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NO: 295, 245, 246, 247, 248 and 249, respectively; (viii) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC and CDR3 LC of SEQ ID NOs: 234, 235, 233, 57, 58 and 241, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC and LCDR3 LC as set forth in one of SEQ ID NOs: 310, 311, 246, 316, 317 and 318, respectively; (ix) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC of SEQ ID NOs: 295, 245, 246, 247, 248, and 249, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC, and LCDR3 LC as set forth in one of SEQ ID NOs: 231, 232, 233, 54, 55, and 240, respectively; or (x) the first CAR comprises CDR1 HC, CDR2 HC, CDR3 HC, CDR1 LC, CDR2 LC, and CDR3 LC of SEQ ID NOs: 310, 311, 246, 316, 317, and 318, respectively, and the second CAR each comprises HCDR1 HC, HCDR2 HC, HCDR3 HC, LCDR1 LC, LCDR2 LC, and LCDR3 LC as set forth in one of SEQ ID NOs: 234, 235, 233, 57, 58, and 241, respectively; and wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, 222 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto. 9. A vector for expressing a CAR molecule, comprising a nucleic acid molecule according to claim 7. 10. The vector according to claim 9, characterized in that the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. 11. A T cell or NK cell for expressing a chimeric antigen receptor (CAR) molecule, wherein the cell comprises the nucleic acid molecule of claim 7. 12. A method for producing a cell comprising transducing a T cell or NK cell using a vector according to claim 9. 13. A method for producing an RNA-engineered cell, comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid molecule according to claim 7, and wherein the cell is a T cell or an NK cell. 14. A pharmaceutical composition for providing antitumor immunity in a subject, wherein the pharmaceutical composition comprises a T cell or NK cell according to any one of claims 1-6 or 11 and a pharmaceutically acceptable carrier. 15. Use of an effective amount of a population of T cells or NK cells comprising a nucleic acid molecule according to claim 7, a CAR molecule according to claim 8, or a vector according to claim 9 or 10; a T cell or NK cell according to any one of claims 1-6 or 11; or a pharmaceutical composition according to claim 14 to provide antitumor immunity in a subject. 16. Application according to paragraph 15, where the subject: (i) a cancer or malignant neoplasm or a precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome or preleukemia, (ii) hematological cancer or solid cancer; (iii) acute leukemia, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, neoplasm plasmacytoid dendritic cells, Waldenstrom's macroglobulinemia, prostate cancer, castration-resistant or therapy-resistant prostate cancer, metastatic prostate cancer, pancreatic cancer, lung cancer, plasma cell proliferative disorder, silent myeloma, smoldering multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytoma, plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, multiple plasmacytoma, systemic amyloidosis in the form of light chain amyloidosis or POEMS syndrome (also known as Crowe-Fukasa syndrome, Takatsuki disease, and PEP syndrome)) or a combination of both; or (iv) multiple myeloma. 17. The use according to claim 15 or 16, wherein the cell or population of T cells or NK cells is formulated for administration in combination with a second therapeutic agent, optionally wherein the second therapeutic agent is selected from: (i) a PD-1 inhibitor, wherein the PD-1 inhibitor is optionally selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224; (ii) a PD-L1 inhibitor, wherein the PD-L1 inhibitor is optionally selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab and BMS-936559; (iii) a LAG-3 inhibitor, wherein the LAG-3 inhibitor is optionally selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280 and REGN3767; (iv) a TIM-3 inhibitor, wherein the TIM-3 inhibitor is optionally selected from the group consisting of MBG453, TSR-022 and LY3321367; (v) a CTLA-4 inhibitor, wherein the CTLA-4 inhibitor is optionally ipilimumab or tremelimumab; (vi) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha subunit (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide, such as hetIL-15; (vii) interleukin-12 (IL-12) polypeptide; or (viii) an mTOR inhibitor, wherein the mTOR inhibitor is optionally RAD001 or rapamycin. 18. A population of T cells or NK cells for expressing a CAR molecule, wherein the population comprises: the first cell population expressing BCMA CAR but not CD19 CAR; a second population of cells expressing the CD19 CAR but not the BCMA CAR; and a third population of cells expressing both BCMA CAR and CD19 CAR; wherein the cells of the first, second and third populations contain a nucleic acid molecule according to paragraph 7. 19. A population of T cells or NK cells for expressing a CAR molecule comprising a nucleic acid molecule according to claim 7, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a third nucleic acid sequence encoding a self-cleavage site. 20. A method for providing antitumor immunity in a subject, comprising administering to the subject an effective amount of a population of T cells or NK cells comprising a nucleic acid molecule according to claim 7, a CAR molecule according to claim 8, or a vector according to claim 9 or 10; a cell according to any one of claims 1-6 or 11; or a pharmaceutical composition according to claim 14. 21. The method according to paragraph 20, wherein the subject: (i) a cancer or malignant neoplasm or a precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome or preleukemia, (ii) hematological cancer or solid cancer; (iii) acute leukemia, B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, neoplasm plasmacytoid dendritic cells, Waldenstrom's macroglobulinemia, prostate cancer, castration-resistant or therapy-resistant prostate cancer, metastatic prostate cancer, pancreatic cancer, lung cancer, plasma cell proliferative disorder, silent myeloma, smoldering multiple myeloma, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytoma, plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, multiple plasmacytoma, systemic amyloidosis in the form of light chain amyloidosis or POEMS syndrome (also known as Crowe-Fukasa syndrome, Takatsuki disease, and PEP syndrome)) or a combination of both; or (iv) multiple myeloma. 22. The method according to claim 20 or 21, wherein the population of T cells or NK cells is formulated for administration in combination with a second therapeutic agent, optionally wherein the second therapeutic agent is selected from: (i) a PD-1 inhibitor, wherein the PD-1 inhibitor is optionally selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224; (ii) a PD-L1 inhibitor, wherein the PD-L1 inhibitor is optionally selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab and BMS-936559; (iii) a LAG-3 inhibitor, wherein the LAG-3 inhibitor is optionally selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280 and REGN3767; (iv) a TIM-3 inhibitor, wherein the TIM-3 inhibitor is optionally selected from the group consisting of MBG453, TSR-022 and LY3321367; (v) a CTLA-4 inhibitor, wherein the CTLA-4 inhibitor is optionally ipilimumab or tremelimumab; (vi) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha subunit (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide, such as hetIL-15; (vii) interleukin-12 (IL-12) polypeptide; or (viii) an mTOR inhibitor, wherein the mTOR inhibitor is optionally RAD001 or rapamycin.

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