TW202216752A - Chimeric molecules providing targeted costimulation for adoptive cell therapy - Google Patents

Abstract

The present invention relates to a chimeric molecule useful in adoptive cell therapy (ACT), and cells comprising the same. The chimeric molecule can act as a modulator of cellular activity enhancing responses when an endogenous T-cell receptor (TCR) is engaged with its cognate antigen. The present invention also provides proteins, nucleic acids encoding the chimeric molecule and therapeutic uses thereof.

Description

Chimeric molecules providing targeted costimulation for adoptive cell therapy

The present invention relates to a chimeric molecule suitable for adoptive cell therapy (ACT) and cells comprising the chimeric molecule. The chimeric molecule can act as a modulator of cellular activity that enhances the response of endogenous T cell receptors (TCRs) to their cognate antigens. The present invention also provides proteins, nucleic acids encoding the chimeric molecules, and therapeutic uses thereof.

Adoptive cell therapy (ACT) using autologous T cells to mediate cancer regression has shown great promise in early clinical trials. Several general approaches have been employed, such as the use of ex vivo expansion of naturally occurring tumor-reactive or tumor-infiltrating lymphocytes (TILs). Additionally, T cells can be genetically engineered to retarget specific tumor antigens. This can be done via gene transfer of peptide (p)-major histocompatibility complex (MHC)-specific T-cell receptors (TCRs) or the interaction of tumor-specific single-chain antibody fragments (scFvs) with T-cell signaling domains such as CD3ζ The T cell signaling domain is called a chimeric antigen receptor (CAR).

TIL and TCR metastasis have proven to be particularly good when targeting melanoma (Rosenberg et al. 2011; Morgan 2006), while CAR therapy has shown great promise in the treatment of certain B-cell malignancies (Grupp et al. 2013).

Costimulatory signals are suitable for achieving stable CAR T cell expansion, function, persistence, and antitumor activity. The success of CAR therapy in leukemia is due in part to the incorporation of costimulatory domains (eg, CD28 or CD137) into CAR constructs, and the signal from the CAR construct synergizes with the signal provided by CD3ζ to enhance antitumor activity. The basis for this observation is the canonical Signal 1/Signal 2 paradigm for T cell activation. Here, signal 1 provided by the TCR complex cooperates with signal 2 provided by co-stimulatory receptors such as CD28, CD137 or CD134 to allow cells to undergo clonal expansion, IL2 production and long-term survival without Signal 1-associated activation-induced cell death (AICD). Furthermore, the involvement of Signal 2 enhances the signal generated via Signal 1, making cells more responsive to low-affinity interactions, such as those encountered during anti-tumor responses.

Targeted costimulation will have beneficial effects on non-CAR-based T cell therapy. For example, the incorporation of costimulatory domains into chimeric TCRs has been shown to enhance T cell responses to pMHC (Govers 2014). While tumor-infiltrating lymphocytes (TILs) utilize their endogenous TCRs to mediate tumor recognition, it is not possible to engineer endogenous TCRs. Thus, TILs are substantially limited because tumor cells express few costimulatory ligands. The ability to induce targeted costimulation of TILs or indeed any other adoptive T cell therapy product would be beneficial.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

The present invention relates to chimeric molecules, particularly chimeric proteins, designed to provide costimulation when an endogenous TCR engages with its cognate antigen. Mechanistically, the proposed construct can be incorporated into the endogenous TCR complex. When the endogenous TCR complex machinery engages its cognate antigen, the TCR receptor complex aggregates, forcing these chimeric constructs to cluster together. This clustering causes activation of its signaling domain, which leads to increased costimulation. This costimulation itself showed measurable improvement in the effector function of recipient T cells: increased activation markers, increased secretion of cytokines (specifically, IL-2) and increased proliferation.

The present invention relates to a chimeric molecule, advantageously a chimeric protein, which provides co-stimulation of T cells when engaging the endogenous T cell receptor. The molecule may comprise a TCR cluster domain and a signaling domain, which may contain a CD40 intracellular domain or a signaling fragment thereof.

The TCR clustering domain may be one or more proteins commonly found in the TCR complex, such as, but not limited to, CD3D, CD3E, CD3G, CD3Z, CD3R-n, and pre-TCRα (PTCRA) TCRα, TCRβ, TCRγ, or TCRδ constant chain.

Messaging domains may also include additional full-length costimulatory domains including, but not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR) or EphB6.

Although CD3D, CD3E, CD3G, and CD3Z act independently, the constructs containing the TCR constant chain (α/β or γ/δ) are preferably co-expressed with their corresponding partners in a bicistronic configuration: TCRα and TCRβ co-express And TCRγ and TCRδ co-express. Thus, constructs containing TCRα advantageously co-express with TCRβ and vice versa; and constructs containing TCRγ should co-express with TCRδ and vice versa. In the case of TILs and any other α-β T cells; the preferred configuration includes TCR γ-δ; and in γ-δ T cells, the preferred configuration includes TCR α-β so that endogenous TCR mechanisms and Interference/disruption of TCR pairing is minimized.

For CD3D, CD3E, CD3G and CD3Z, the transmembrane and extracellular portions are preferably utilized. However, the present invention also encompasses some or all of the intracellular components that can potentially minimize disruption of endogenous TCR complex signaling or facilitate further amplification of endogenous TCR signaling.

In another aspect, the present invention provides a chimeric protein comprising a clustering domain and a signaling domain, the signaling domain may contain a CD40 intracellular domain or a signaling fragment thereof. In certain embodiments, the clustering domains are capable of oligomerization and/or self-assembly. In certain embodiments, clustering comprises forming homodimers or homotrimers. In certain embodiments, clustering comprises oligomerization using different proteins to form heterodimers or heterotrimers. In certain embodiments, the chimeric protein is constitutive in signaling, eg, independent of receptor engagement by extracellular ligands or independent of extracellular ligands attached to different cells Receptor engagement. In certain embodiments, the clustering domain comprises a transmembrane domain. In certain embodiments, the clustering domain comprises a transmembrane domain and further comprises an activating mutation that promotes dimerization or oligomerization. In certain embodiments, the clustering domain comprises an extracellular domain, such as, but not limited to, an extracellular domain of a receptor. In certain embodiments, the clustering domain comprises the extracellular domain of the receptor and further comprises an activating mutation in the extracellular domain that promotes dimerization or oligomerization. In certain embodiments, the clustering domain comprises a leucine zipper. In certain embodiments, the leucine zipper comprises or constitutes a transmembrane domain. In certain embodiments, the leucine zipper comprises or constitutes a soluble domain. Non-limiting examples of clustering domains include thrombopoietin receptor (TpoR), erythropoietin receptor (EpoR), growth hormone receptor (GHR), glycophorin A (GPA) transmembrane domains and activating mutants thereof the cluster domain. In certain embodiments, clustering can be modulated by small molecules. In certain embodiments, clustering can be modulated by post-translational modification.

In another aspect, the invention provides a chimeric protein comprising an extracellular ligand binding domain linked to an intracellular signaling domain via a transmembrane domain. In certain embodiments, the extracellular ligand binding domain is selected or engineered to bind to an extracellular ligand that holds two or more copies of the chimeric protein in close proximity to each other to Causes the messaging field to be activated. The extracellular ligand binding domain is considered part of a specific binding pair (sbp) and the extracellular ligand is the second part of the specific binding pair. In certain embodiments, one member of the sbp comprises a protein or receptor or extracellular portion thereof and the second sbp comprises a binding protein specific for the first member of the sbp. In certain embodiments, the extracellular sbp is bivalent. In certain embodiments, the extracellular sbp is trivalent. Non-limiting examples of extracellular ligands include antibodies and bivalent antigen-binding fragments thereof. Non-limiting examples of extracellular ligand binding domains of chimeric proteins of the invention (ie, sbp members) include, but are not limited to, NKG2A, CD27, CD137, GITR, PD-1, PD-L1, FasL, OX40 , CTLA4, ICOS, CD40, EGFR, HER2 and their extracellular fractions. Complementary sbp members include (but are not limited to): pembrolizumab for PD1, trastuzumab for HER2, cetuximab for EGFR, tramezumab for CTLA4 ( tremelimumab), varlilumab for CD27, and urelumab for CD137. In certain embodiments, the intracellular signaling domain comprises a CD40 intracellular domain or a signaling fragment thereof.

In certain embodiments of the invention, the CD40 signaling domain comprises SEQ ID NO:154, SEQ ID NO:155, or SEQ ID NO:156. In certain embodiments, the CD40 messenger fragment comprises, consists of, or consists essentially of the SH3 motif (KPTNKAPH, PNKAPHP or PNKAPH), the TRAF2 motif (PKQE, PKQET, PVQE, PVQET, SVQE, SVQET) , a TRAF6 motif (QEPQEINFP or QEPQEINFP), a PKA motif (KKPTNKA, SRISVQE), or a combination thereof, or a full-length CD40 intracellular domain. In certain embodiments, one or more of the SH3, TRAF2, TRAF6, or PKA motifs of the CD40 messaging domain are mutated. In certain embodiments, one or more of the SH3, TRAF2, TRAF6, or PKA motifs of the CD40 messaging domain are present in multiple copies.

Accordingly, it is an object of the present invention to exclude any previously known product, process for making the product, or method of using the product within the present invention, so that the applicant reserves the right to hereby disclose any previously known product, process, or method. Disclaimer. It should also be noted that the present invention is not intended to cover within the scope of the present invention any product, manufacture of such product that does not comply with the written description and implementation requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Chapter 83 of the EPC). process or method of using the product, the applicant reserves the right to hereby disclose any disclaimer of any previously described product, the process of making the product, or the method of using the product. It may be advantageous to follow Art. 53(c) EPC and Rule 28(b) and (c) EPC in the practice of the present invention. All rights that are expressly reserved for any embodiment of the subject matter of any one or more of the applicants in the pedigree of this application or in any other pedigree or any previously filed application of any third party are expressly reserved. . Nothing in this article should be taken as a commitment.

It should be noted that in this disclosure, and particularly in the claims and/or paragraphs, terms such as "comprises/comprised/comprising" and the like may have the meanings ascribed to them under US patent law; for example, they may means "includes/included/including" and the like; and terms such as "consisting essentially of/consists essentially of" have the meaning ascribed to them in US patent law, eg Elements that are not explicitly recited are permitted to exclude elements found in the prior art or which affect the essential or novel characteristics of the invention.

These and other embodiments are disclosed in or apparent from and encompassed by the following description.

RELATED APPLICATIONS AND INCORPORATED BY REFERENCE

This application claims priority to US Patent Application Serial No. 63/053,498, filed on July 17, 2020, and US Patent Application Serial No. 63/222,916, filed on July 16, 2021, the contents of which are as follows: Incorporated herein by reference in its entirety.

Refer to GB Patent Application No. 1900858.0 filed on January 22, 2019, US Patent Application No. 62/951,770 filed on December 20, 2019, and International Application PCT/GB2020/ filed on January 20, 2020 050120 and US Provisional Patent Application 63/053,494, filed July 17, 2020.

The aforementioned applications, and all documents cited therein or during the prosecution of them ("Application citations"), and all documents cited or referenced in the application citations, and all documents cited or referenced herein ( "Documents cited herein"), and all documents cited or referenced in the documents cited herein, and any manufacturer's instructions, descriptions, products of any product mentioned herein or in any document incorporated by reference herein Specifications and product descriptions are hereby incorporated by reference and may be used in the practice of the present invention. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

As used herein, "full-length protein" or "full-length receptor" refers to a receptor protein, such as the CD40 receptor protein. The term "full-length" encompasses receptor proteins which, upon cleavage of their signal peptide, lack up to about 5 or up to 10 amino acids at the N-terminus of the mature receptor protein, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. For example, although the specific cleavage site for the receptor N-terminal signal peptide can be defined, variability in the exact cleavage site has been observed. The term "full length" does not imply the presence or absence of amino acids of the receptor's N-terminal signal peptide. In one embodiment, the term "full-length" (eg, full-length CD28 or full-length CD40 intracellular domain according to certain aspects of the invention) encompasses mature receptor proteins that lack an N-terminal signal peptide (eg, according to certain aspects of the invention) (like CD28), once its signal peptide has been cleaved, it lacks up to about 5, eg, 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminus of the mature receptor protein. As mentioned above, a "full-length" CD28 receptor or other receptor or TCR cluster domain according to various aspects of the invention does not include a signal peptide and is N-terminal to the mature receptor protein (eg N-terminal residues N, Up to about 5, eg, 1, 2, 3, 4, 5, or up to 10 amino acids may be absent at K, I, L and/or V). This is shown in exemplary fusions (eg SEQ ID No. 4-12) (it should be noted that these fusions may lack up to about 5 at the N-terminus of the mature receptor protein, eg 1, 2, 3, 4 , 5 or up to 10 amino acids, as indicated in the boxed regions).

The chimeric protein of the present invention may comprise a TCR clustering domain and a signaling domain, which may advantageously comprise a CD40 intracellular domain.

The TCR clustering domain may advantageously comprise one or more proteins normally found in the TCR complex. Advantageously, the TCR clustering domain may comprise CD3D, CD3E, CD3G or CD3Z or parts thereof.

The CD3D antigen delta polypeptide (TiT3 complex) encoded by the CD3D gene is part of the T cell receptor/CD3 complex (TCR/CD3 complex) and is involved in T cell development and signal transduction. The encoded membrane protein represents the delta subunit of the CD3 complex and, along with four other CD3 subunits, binds TCR alpha/beta or TCR gamma/delta to form the TCR/CD3 complex on the T cell surface. Defects in this gene are the cause of somatic recessive T-cell-negative/B-cell-positive/NK-cell-positive severe combined immunodeficiency disease (SCIDBNK). Two transcript variants of this gene have been found encoding two different isoforms. Other variants may also exist, but the full-length nature of their transcripts has yet to be defined. See eg annotation release 109.20200522 Assembly GRCh38.p13 (GRCh38.p13).

The CD3E antigen ε polypeptide (TiT3 complex) encoded by the CD3E gene is the CD3-ε polypeptide, which together with CD3-γ, CD3-δ and CD3-ζ forms heterodimers with T cell receptors α/β and γ/δ T cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signaling pathways. The genes encoding the epsilon, gamma and delta polypeptides are located in the same cluster on chromosome 11. ε polypeptides play an important role in T cell development. See, eg, Accession Nos. NM_000733 and NM_007648 for mRNA sequences, and Accession Nos. NP_000724 and NP_031674 for protein sequences.

The CD3G antigen gamma polypeptide (TiT3 complex) is encoded by the CD3G gene. CD3G (gamma chain) is one of the four peptides (gamma, delta, epsilon and zeta) that form CD3. Deficiencies in CD3G are associated with T cell immunodeficiency. See, eg, Accession Nos. NM_000073 and NM_009850 for mRNA sequences, and Accession Nos. NP_000064 and NP_033980 for protein sequences.

The CD3Z antigen zeta polypeptide (TiT3 complex) is encoded by CD3Z. The glycoprotein CD3 zeta chain is also known as the T cell receptor T3 zeta chain or CD247 (cluster of differentiation 247). T cell receptor ζ (ζ) and T cell receptor α/β and γ/δ heterodimers and CD3-γ, CD3-δ and CD3-ε form a T cell receptor-CD3 complex ξ chain in the antigen Recognition plays an important role in coupling to several intracellular signaling pathways. Low expression of the antigen leads to a weakened immune response. Two alternatively spliced transcript variants of this gene encoding different isoforms have been found. CD247 has been shown to interact with Janus kinase 3 and the homolog of the protein unc-119.關於mRNA序列參見例如寄存編號NM_000734、NM_198053、NM_001378515、NM_001378516、NM_001113391、NM_001113392、NM_001113393、NM_001113394及NM_031162，且關於蛋白質序列參見寄存編號NP_000725、NP_932170、NP_001365444、NP_001365445、NP_112439.1、NP_001106862.1、NP_001106862、 NP_001106863 and NP_001106864.

CD3 n (n) is the alternative splicing product of the gene encoding CD3 zeta (Clayton et al 1991 Proceedings of the National Academy of Sciences of the USA 88, 5202-5206) and has an apparent molecular weight of 23 kDa (Orloff et al 1989 Journal of Biological Chemistry 264, 14812-14817). CD3 zeta is primarily found in a homodimeric form, but a portion (about 10%) of CD3 zeta was found to complex with CD3 zeta to form a heterodimer and this form is thought to confer distinct signaling properties (Orloff et al. 1989 Journal of Biological Chemistry 264, 14812-14817).

The TCR clustering domain may comprise the invariant chain of TCRα (TCRα), TCRβ (TCRβ), TCRγ (TCRγ) or TCRδ (TCRδ), or a portion thereof. Representative loci information is provided below: T cell receptor alpha locus identifier symbol TRA Alt symbol TCRA, TRA@ NCBI gene 6955 HGNC 12027 OMIM 186880 other information locus Chr. 14q11.2 T cell receptor beta locus identifier symbol TRB Alt. symbol TCRB, TRB@ NCBI gene 6957 HGNC 12155 OMIM 186930 other information locus Chr. 7q34 T cell receptor delta locus identifier symbol TRD Alt. symbol TCRD, TRD@, TCRDV1 NCBI gene 6964 HGNC 12252 other information locus Chr. 14q11.2 T cell receptor gamma locus identifier symbol TRG Alt. symbol TCRG, TRG@ NCBI gene 6965 HGNC 12271 other information locus Chr. 7 p14

The term "T cell receptor" or "TCR" refers to a heterodimeric receptor composed of paired αβ or γδ chains on the surface of T cells. Each alpha, beta, gamma and delta chain is composed of two Ig-like domains: a variable domain (V) conferring antigen recognition via complementarity determining regions (CDRs), followed by anchoring via linker peptide and transmembrane (TM) regions to the constant domain (C) of the cell membrane. The TM region is combined with the constant subunit of the CD3 messaging device. Each of the V domains has three CDRs. Complex interactions between these CDRs and antigenic peptides bound to proteins encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) ) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, p. 868).

Costimulatory receptor receptor proteins suitable for use in the chimeric proteins of the present invention include, but are not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR) or EphB6, which contain the extracellular ligand binding domain in their native form and intracellular signal transduction domains. For example, CD2 is characterized as a cell adhesion molecule found on the surface of T cells and capable of initiating intracellular signals required for T cell activation. CD27 is characterized as a type II transmembrane glycoprotein belonging to the TNFR superfamily (TNFRSF) whose expression on B cells is induced by antigen receptor activation in B cells. CD28 is a protein on T cells and is the receptor for CD80 (B7.1) and CD86 (B7.2) ligands on antigen presenting cells. CD137 (4-1BB) ligands are found on most leukocytes and some non-immune cells. OX40 ligands are expressed on many antigen presenting cells such as DC2 (dendritic cells), macrophages and B lymphocytes. In one embodiment, the costimulatory receptor protein is full-length CD28 as defined herein.

CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and several isoforms are produced by alternative splicing. Its ligand CD154 (also known as CD40L) is a protein predominantly expressed on activated T cells. For reference, the human CD40 isoform 1 protein sequence is contained in GenBank Accession No. NP_001241.1, including the signal peptide (amino acids 1-20), the transmembrane domain (amino acids 194-215), and the cytoplasmic domain (amino acids 216-277) (SEQ ID NO: 22). CD40 receptor signaling involves adaptor proteins, including but not limited to TNF receptor-associated factor (TRAF), and the cytoplasmic domain contains signaling components, including but not limited to SH3 motif (KPTNKAPH), TRAF2 motif (PKQE, PVQE, SVQE), TRAF6 motif (QEPQEINFP) and PKA motif (KKPTNKA, SRISVQE). Additional motifs that bind to TRAF1, TRAF2, TRAF3, and TRAF5 include the major consensus sequence (P/S/A/T)X(Q/E)E or the minor consensus sequence PXQXXD, and are identifiable from TNFR family members such as But not limited to CD30, Ox40, 4-1BB and EBV oncogenic protein LMP1) or obtained therefrom. (See e.g. Ye, H et al., The Structural Basis for the Recognition of Diverse Receptor Sequences by TRAF2 . Molecular Cell, 1999; 4(3):321-30. doi: 10.1016/S1097-2765(00)80334-2; Park HH, Structure of TRAF Family: Current Understanding of Receptor Recognition . Front. Immunol. 2018; 9:1999. doi: 10.3389/fimmu.2018.01999).

The examples disclosed herein demonstrate the operation of CD40 as a signaling domain and further demonstrate that the interleukin and chemokine performance profiles are altered by signaling domain selection. In this regard, the CD40 signaling domains of the present invention provide distinct and overlapping responses induced by distinct factor binding sites. (See eg, Ahonen, CL et al., The CD40-TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells . Nat Immunol. 2002; 3: 451-456; Mackey MF et al., Distinct contributions of different CD40 TRAF binding sites to CD154-induced dendritic cell maturation and IL-12 secretion . Eur J Immunol. 2003; 33: 779-789; Mukundan et al., TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages . J Immunol. 2005; 174: 1081-1090).

In certain embodiments, the chimeric proteins of the invention comprise substantially all of the CD40 costimulatory domain. In certain embodiments, the chimeric proteins of the invention comprise two or more CD40 costimulatory domains. In certain embodiments, the chimeric proteins of the invention comprise CD40 costimulatory domain signaling components or motifs, including but not limited to SH3 motif (KPTNKAPH), TRAF2 motif (PKQE, PVQE, SVQE), TRAF3 motif, TRAF6 motif (QEPQEINFP) or PKA motif (KKPTNKA, SRISVQE) and two or more, or three or more, or four or more such components or motifs, which may in multiple replicas and in any order. In certain embodiments, the chimeric proteins of the invention comprise a CD40 costimulatory domain and a CD40 costimulatory domain signaling component or motif.

In certain embodiments, the selection of one or more costimulatory domain signaling components or motifs is determined by the desired costimulatory activity of the cell in which the chimeric protein is expressed and/or more closely associated with the signaling components or motifs, Or avoid co-stimulatory activities more closely related to signaling components or motifs.

In certain embodiments, in addition to the CD40 costimulatory domain, or a signaling component or motif thereof, or two or more such domains or components or motifs, or a combination thereof, the chimeric protein signaling domain also includes Another full-length costimulatory domain or signaling component thereof, derived from, but not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99 , CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR) or EphB6.

For reference, the human CD28 protein sequence is contained in GenBank Accession No. NP_006130.1, including the signal peptide (amino acids 1-18), the extracellular domain (amino acids 19-152), the transmembrane domain (amino acids 153) -179) and the cytoplasmic domain (amino acids 180-200). The extracellular domain includes an immunoglobulin-type domain (amino acids 21-136), which contains amino acids that constitute the antigen binding site and amino acids that form a homodimer interface. The extracellular domain includes several asparagine residues that can be glycosylated, and the intracellular domain includes phosphorylated serine and tyrosine residues.

For reference, the human CD8 alpha chain protein sequence is contained in GenBank Accession No. NP_001139345.1, including signal peptide (amino acids 1-21), extracellular domain (amino acids 22-182), transmembrane domain (amino acid 183) -203) and the cytoplasmic domain (amino acids 204-235). The extracellular domain includes an immunoglobulin-type domain (amino acids 28-128), which contains amino acids that constitute the antigen-binding site and amino acids that form a homodimer interface. The extracellular domain includes several asparagine residues that can be glycosylated, and the intracellular domain includes phosphorylated serine and tyrosine residues.

For reference, the human IgG4 constant region sequences are contained in UniProtKB/Swiss-Prot: Accession No. P01861.1, including CH1 (amino acids 1-98), hinge (amino acids 99-110), CH2 (amino acids 99-110) acid 111-220), CH3 (amino acid 221-327). The CH2 region includes asparagine at amino acid 177, which is glycosylated and associated with Fc receptors and antibody-dependent cell-mediated cytotoxicity (ADCC).

For reference, the protein sequence of human CD137 (41BB), another TNFR superfamily member, is contained in GenBank Accession No. NP_001552.2, including the signal peptide (amino acids 1-23), the extracellular domain (amino acids 24-186 ), the transmembrane domain (amino acids 187-213), and the cytoplasmic domain (amino acids 214-255).

For reference, the human CD134 (OX40) protein sequence is contained in GenBank Accession No. NP_003318.1, including the signal peptide (amino acids 1-28), the extracellular domain (amino acids 29-214), the transmembrane domain (amino acids 29-214) amino acids 215-235) and the cytoplasmic domain (amino acids 236-277). This receptor has been shown to activate NF-κB through its interaction with the adaptor proteins TRAF2 and TRAF5, and studies have shown that this receptor promotes the expression of apoptosis inhibitors BCL2 and BCL21L1/BCL2-XL.

The human T cell surface antigen CD2 has at least two isoforms. For reference, the human CD2 isoform 1 protein sequence is contained in NP_001315538.1, including the signal peptide (amino acids 1-24), the extracellular domain (amino acids 25-235), the transmembrane domain (amino acids 25-235) acid 236-261) and the cytoplasmic domain (amino acids 262-377). The human CD2 isoform 2 protein sequence is contained in NP_001758.2.

For reference, the human CD357 (GITR) isoform-1 protein sequence is contained in GenBank Accession No. NP_004186.1, including the signal peptide (amino acids 1-25), the extracellular domain (amino acids 26-162) , the transmembrane domain (amino acids 163-183) and the cytoplasmic domain (amino acids 184-241).

For reference, the human CD29 (β1 integrin) protein sequence is contained in GenBank Accession No. NP_596867, including the signal peptide (amino acids 1-20), the extracellular domain (amino acids 21-728), the transmembrane domain (amino acids 21-728) amino acids 729-751) and the cytoplasmic domain (amino acids 752-798).

The human CD150 (SLAM) protein sequence has several isoforms. In addition to the transmembrane form of CD150 (mCD150), cells of the hematopoietic lineage express mRNA encoding the secreted form of CD150 (sCD150), which lacks the entire transmembrane region of 30 amino acids. For reference, the human SLAM isoform b is contained in GenBank Accession No. NP_003028.1, including the signal peptide (amino acids 1-20), the extracellular domain (amino acids 21-237), the transmembrane domain (amino acids 21-237) acid 238-258) and the cytoplasmic domain (amino acid 259-335). The human SLAM isoform a is contained in GenBank Accession No. NP_001317683.1.

In embodiments of the invention, the chimeric protein can be expressed alone under the control of a promoter in a therapeutically active population of therapeutic cells, such as tumor infiltrating lymphocytes (TIL). Alternatively, the chimeric protein can be expressed in conjunction with a therapeutically transgenic gene, such as a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). Suitable TCRs and CARs are well known in the literature, such as HLA-A*02-NYESO-1 specific TCRs (Rapoport et al. Nat Med 2015) or anti-CD19scFv.CD3ζ fusion CARs (Kochenderfer et al. J Clin Oncol 2015) , which have been successfully used to treat myeloma or B-cell malignancies, respectively. The chimeric proteins described herein can be expressed with any known CAR or TCR, thereby providing cells with tunable growth switches to allow cell expansion in vitro or in vivo, and provide TCR or CAR for anticancer activity Mechanisms of learned activation of form. Accordingly, the present invention provides a cell for adoptive cell therapy comprising a chimeric protein as described herein and a TCR and/or CAR that specifically binds to a tumor-associated antigen. Exemplary chimeric proteins comprising CD28 include the extracellular antigen binding domain as well as the extracellular transmembrane and intracellular signaling domains.

The chimeric proteins of the present invention optionally comprise a spacer region between the TCR clustering domain and the costimulatory receptor. As used herein, the term "spacer" refers to the extracellular domain of a chimeric protein that separates the TCR clustering domain from the signaling domain of the chimeric protein. In certain embodiments, long spacers are employed, eg, targeting juxtamembrane epitopes or glycosylated antigens (see Guest R.D. et al., The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J. Immunother. 2005;28:203-211; Wilkie S. et al., Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor. J. Immunol. 2008;180 :4901-4909). In other embodiments, the chimeric protein has a short spacer, e.g., to target far membrane epitopes (see Hudecek M. et al., Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013;19:3153-3164; Hudecek M. et al., The nonsignalling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol. Res. 2015;3:125-135). In certain embodiments, the spacer comprises all or a portion of an IgG hinge or is derived from an IgG hinge, including but not limited to IgGl, IgG2, or IgG4. "Derived from an Ig hinge" means comprising an inserted, deleted or mutated spacer in the IgG hinge. In certain embodiments, the spacer may comprise all or a portion of one or more antibody constant domains, such as, but not limited to, CH2 and/or CH3 domains. In certain embodiments, in the spacer comprising all or a portion of the CH2 domain, the CH2 domain is modified so as not to bind to the Fc receptor. For example, Fc receptor binding in myeloid cells has been found to impair CAR T cell functionality. In certain embodiments, the spacer comprises all or a portion of an Ig-like hinge from CD28, CD8, or other proteins comprising a hinge region. In certain embodiments of the invention comprising spacers, the spacers are 1 and 50 amino acids in length.

In certain embodiments, the chimeric protein extracellular domain comprises a linker. Linkers comprise short-running amino acids used to link domains (eg, binding domains) to spacers or transmembrane domains. For flexibility in binding the ligand, the ligand binding domain will typically be attached to the spacer or transmembrane domain by a flexible linker comprising about 5 to 25 amino acids, such as AAAGSGGSG or GGGGSGGGGSGGGGS. In certain embodiments, the chimeric protein comprises a TCR clustering domain joined directly to the signaling domain by a linker and without a spacer. In certain embodiments, the chimeric protein comprises a binding domain directly joined to a transmembrane via a spacer and without a linker.

As discussed above, in certain embodiments, the chimeric protein comprises a full-length primary costimulatory receptor, which can include, but is not limited to, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46 , CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR) or extracellular ligands of EphB6 Body binding part and intracellular signaling part. In other embodiments, the chimeric protein may comprise, for example, the extracellular ligand binding domain of one of the foregoing proteins and the intracellular signaling domain of another of the foregoing proteins. In certain embodiments, the signaling portion of the chimeric protein comprises a single signaling domain. In other embodiments, the signaling portion of the chimeric protein comprises a second intracellular signaling domain such as (but not limited to): CD2, CD27, CD28, CD40, CD134 (OX40), CD137 (4-1BB), CD150 (SLAM ). In certain embodiments, the first and second intracellular signaling domains are the same. In other embodiments, the first and second intracellular signaling domains are different. In certain embodiments, costimulatory receptors are capable of dimerization. Without being bound by theory, it is believed that the chimeric protein dimerizes or associates with other accessory molecules to initiate the signal. In certain embodiments, the chimeric protein dimerizes or associates with accessory molecules via transmembrane domain interactions. In certain embodiments, dimerization or association with an accessory molecule is aided by co-stimulatory receptor interactions in the intracellular portion and/or the extracellular portion of the co-stimulatory receptor.

Although the primary function of the transmembrane is to anchor the chimeric protein in the T cell membrane, in certain embodiments, the transmembrane domain affects the function of the chimeric protein. In certain embodiments, the transmembrane domain consists of a full-length primary costimulatory receptor domain. In embodiments of the invention wherein the chimeric protein construct comprises the extracellular domain of one receptor and the intracellular signaling domain of a second receptor, the transmembrane domain may be the transmembrane domain of the extracellular domain or the intracellular domain. In certain embodiments, the transmembrane domain is from CD4, CD8α, CD28 or ICOS. Related use of ICOS transmembrane domain with increased CAR T cell persistence and overall antitumor efficacy by Gueden et al. (Guedan S. et al. Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight. 2018;3 :96976). In one embodiment, the transmembrane domain comprises a hydrophobic alpha helix that spans the cell membrane.

In some embodiments, amino acid sequence variants of the TCR cluster domain portions or other portions provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the moiety. Amino acid sequence variants of the antibody moiety can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the cluster moiety or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of an antibody portion. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics.

In some embodiments, TCR clustering domain portions comprising one or more amino acid substitutions, deletions or insertions are provided. Amino acid substitutions can be introduced into the binding domain of interest and the product screened for a desired activity (eg, maintained/improved clustering or reduced immunogenicity). In certain embodiments, amino acid substitutions can be introduced into one or more of the primary costimulatory receptor domain (extracellular or intracellular), the secondary costimulatory receptor domain, or the extracellular co-receptor domain. Accordingly, the present invention encompasses the chimeric proteins and components specifically disclosed herein, as well as variants thereof, ie, chimeric proteins and components having at least 75%, at least 80%, at least 85% of the amino acid sequences specifically disclosed herein , at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. The terms "similarity identity", "percent identity" and "percent homology" are used when referring to specific sequences, as described in the University of Wisconsin GCG software program BestFit, other algorithms such as BLAST, psiBLAST may be used or TBLASTN (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448).

A particular amino acid sequence variant may differ from the reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5 to 10, 10 to 20, or 20 to 30 amino acids. In some embodiments, a variant sequence may comprise a reference sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more inserted, deleted or substituted residues. For example, 5, 10, 15, up to 20, up to 30, or up to 40 residues may be inserted, deleted, or substituted.

In some preferred embodiments, the variant may differ from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. Conservative substitutions involve replacing an amino acid with a different amino acid with similar properties. For example, an aliphatic residue can be replaced by another aliphatic residue, a non-polar residue can be replaced by another non-polar residue, an acidic residue can be replaced by another acidic residue, a basic residue can be replaced by another Another basic residue is replaced, a polar residue can be replaced by another polar residue or an aromatic residue can be replaced by another aromatic residue. Conservative substitutions can be made, for example, between amino acids within the following groups:

Conservative substitutions are shown in the table below. original residue Exemplary substitution better replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu Leu (L) n-leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu

Amino acids can be divided into different categories according to the common side chain properties: a. Hydrophobicity: ortholeucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln ; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues affecting chain orientation: Gly, Pro; Aromatic: Trp, Tyr, Phe. Non-conservative substitutions will result in members of one of these classes being exchanged for another class. The cells used in the present invention can be any lymphocytes suitable for adoptive cell therapy, such as T cells or natural killer (NK) cells, NKT cells, gamma/delta T cells or T regulatory cells. The cells can be allogeneic or autologous to the patient.

T cells or T lymphocytes are the type of lymphocytes that play a central role in cell-mediated immunity. It can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on the cell surface. There are various types of T cells, as outlined below. Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 molecules at their surface.

These cells recognize their targets by binding to antigens associated with MHC class I present on the surface of all nucleated cells. Via IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be deactivated to an anergic state, which prevents autoimmune diseases, such as experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persist long after the infection has resolved. They rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigens, thus providing the immune system with a "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO. Regulatory T cells (Treg cells), formerly known as suppressor T cells, are critical for maintaining immune tolerance. Its main role is to shut down T cell-mediated immunity to the end of the immune response and suppress actively reactive T cells that escape the negative selection process in the thymus.

Two main classes of CD4+ Treg cells have been described - naturally occurring Treg cells and adaptive Treg cells. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) are produced in the thymus and are closely related to the production of T cells and myeloid (CD11c ⁺ ) and plasmacytoid (CD123 ⁺ ) dendritic cells that have been activated with TSLP interactions between them. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can be generated during normal immune responses.

Natural killer cells (or NK cells) are a type of cytolytic cell that forms part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute a third type of cell distinct from the common lymphoid precursor cells that give rise to B and T lymphocytes.

In certain embodiments, the therapeutic cells of the invention comprise autologous cells engineered to express the chimeric protein. In certain embodiments, the therapeutic cells of the invention comprise allogeneic cells engineered to express the chimeric protein. Autologous cells expressing chimeric proteins can advantageously avoid graft-versus-host disease (GVHD) due to TCR-mediated recognition of recipient alloantigens.

One aspect of the present invention provides nucleic acid sequences of the present invention that encode any of the chimeric proteins, polypeptides or proteins described herein (including functional portions and functional variants thereof). As used herein, the terms "polynucleotide", "nucleotide" and "nucleic acid" are intended to be synonymous with each other. The skilled artisan will appreciate that, due to the degeneracy of the genetic code, a large number of different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it will be appreciated that the skilled artisan can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide will be expressed (eg codon optimization). Nucleic acids according to the present invention may comprise DNA or RNA. It can be single-stranded or double-stranded. It can also be a polynucleotide that includes synthetic or modified nucleotides therein. Many different types of modifications to oligonucleotides are known in the art. Such modifications include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is understood that polynucleotides can be modified by any method available in the art. Such modifications can be made in order to enhance the in vivo activity or longevity of the polynucleotide of interest.

The terms "variant", "homolog" or "derivative" in reference to a nucleotide sequence include any substitution, change, modification, substitution, deletion or addition from or to one (or more) nucleic acid of the sequence.

The nucleic acid sequence may encode the protein sequence set forth in SEQ ID NO: 2 or a variant thereof. The nucleotide sequence may comprise a codon-optimized nucleic acid sequence shown to be engineered for expression in human cells.

The invention also provides nucleic acid sequences comprising a nucleic acid sequence encoding a chimeric protein and another nucleic acid sequence encoding a T cell receptor (TCR) and/or a chimeric antigen receptor (CAR).

Nucleic acid sequences can be joined by sequences that allow the co-expression of two or more nucleic acid sequences. For example, the construct may comprise an internal promoter, an internal ribosomal entry sequence (IRES), or a sequence encoding a cleavage site. The cleavage site is self-cleavable so that when a polypeptide is produced, it does not require any external cleavage activity to be cleaved into discrete proteins. Various self-cleaving sites are known, including foot and mouth disease virus (FMDV) and the 2A self-cleaving peptide. The co-expressing sequence may be an internal ribosome entry sequence (IRES). The co-expressed sequence can be an internal promoter.

In one aspect, the present invention provides a vector comprising a nucleic acid sequence or nucleic acid construct of the present invention.

Such vectors can be used to introduce nucleic acid sequences or nucleic acid constructs into host cells such that they express one or more chimeric proteins according to the first aspect of the invention and optionally one or more other proteins of interest (POIs) , such as TCR or CAR. The vector may be, for example, a plastid or viral vector, such as a retroviral or lentiviral vector, or a transposon-based vector or synthetic mRNA.

The nucleic acids of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety.

Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of one or more transgenic genes and their dissemination in daughter cells. The vector may be capable of transfecting or transducing lymphocytes including T cells or NK cells. The present invention also provides vectors into which the nucleic acid of the present invention is inserted. Expression of natural or synthetic nucleic acids encoding chimeric proteins and, optionally, TCRs or CARs is typically accomplished by operably linking nucleic acids encoding chimeric proteins and TCR/CAR polypeptides or portions thereof to one or more promoters and constructing body is incorporated into the expression vector to achieve.

Other promoter elements, such as enhancers, regulate transcription initiation frequency. Typically, these elements are located in a region 30-110 bp upstream of the initiation site, but several promoters have recently been shown to contain functional elements that are also downstream of the initiation site. The spacing between promoter elements is generally flexible such that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high expression levels of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) ) promoter, MoMuLV promoter, MSCV promoter, MND promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as (but not Limited to) actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter.

The vector may be suitable for replication and integration in eukaryotic host cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters suitable for regulating the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology, see also WO 01 /96584; WO 01/29058; and US Patent No. 6,326,193. In some embodiments, the represented constructs are shown in SEQ ID NOs: 32-65 and 67-79. In some embodiments, the nucleic acid is a polycistronic construct that allows expression of multiple transgenic genes (eg, chimeric proteins and TCR and/or CAR, etc.) under the control of a single promoter. In some embodiments, the transgenic genes (eg, chimeric proteins and TCR and/or CAR, etc.) are separated by a self-cleaving 2A peptide. Examples of 2A peptides suitable for use in nucleic acid constructs of the present invention include F2A, P2A, T2A, and E2A. In other embodiments of the present invention, the nucleic acid constructs of the present invention are polycistronic constructs comprising two promoters; one promoter driving chimeric protein expression and the other promoter driving TCR or CAR expression. In some embodiments, the dual promoter constructs of the present invention are unidirectional. In other embodiments, the dual promoter constructs of the present invention are bidirectional. To assess the performance of the chimeric protein polypeptide or portion 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 identification and Select expressing cells.

A source of cells, eg, immune effector cells (eg, T cells or NK cells), is obtained from the subject prior to expansion and genetic modification. The term "subject" is intended to include living organisms (eg, mammals) that can elicit an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, eg, by elutriation via PERCOLL ^™ gradient centrifugation or by countercurrent centrifugation.

In another aspect, tumor-infiltrating cells (TILs) are isolated and/or expanded from tumors, eg, by disrupted, dissected, or enzymatically digested tumor biopsies or clumps.

Specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells can be further isolated by positive or negative selection techniques. For example, in one aspect, T cell lines are incubated with anti-CD3/anti-CD28 conjugated beads (such as DYNABEADS® M-450 CD3/CD28 T) for a period of time sufficient to positively select desired T cells to separate. In one aspect, the time period is about 30 minutes. In another aspect, the period of time ranges from 30 minutes to 36 hours or more and all integer values therebetween. In another aspect, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation period is 24 hours. Longer incubation times can be used to isolate T cells in any situation where very few T cells are present compared to other cell types, such as isolation of tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. In addition, the use of longer incubation times can increase the efficiency of capturing CD8+ T cells. Thus, by simply shortening or prolonging the time to allow T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as further described herein), for or against at the initiation of culture or Subpopulations of T cells may be preferentially selected at other time points during the method. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, a subset of T cells can be preferentially selected for or against at the initiation of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection may also be used in the context of the present invention. In some aspects, a selection procedure may be required and "unselected" cells are used in the activation and expansion process. "Unselected" cells may also undergo other rounds of selection.

Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is to sort and/or select cells via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150, and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti-CD25-conjugated beads or other similar selection methods.

The methods described herein can include selection of specific subsets of immune effector cells (eg, T cells, CD25+ depleted cells as a T regulatory cell depleted population), eg, using negative selection techniques such as those described herein. Preferably, the T-regulatory depleted cell population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

Specific subsets of chimeric protein effector cells that specifically bind to the antigen of interest can be enriched by positive selection techniques. For example, in some embodiments, the effector cell line is enriched by incubating with the target antigen-binding beads for a period of time sufficient to positively select the desired abTCR effector cells. In some embodiments, the time period is about 30 minutes. In some embodiments, the period of time is in the range of 30 minutes to 36 hours or longer, including all ranges between such values. 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 10 to 24 hours. In some embodiments, the incubation period is 24 hours. With regard to the isolation of effector cells present at low levels in heterogeneous cell populations, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate effector cells in any situation where fewer effector cells are present compared to other cell types. Those skilled in the art will recognize that multiple rounds of selection may also be used in the context of the present invention.

T cells used for stimulation can also be frozen after washing steps. After a wash step to remove plasma and platelets, cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and will be suitable for this situation, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or 10% polydextrose 40 and 5% dexamethasone. Spinose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% polydextrose 40 and 5% dextrose, 20% human serum albumin Protein and 7.5% DMSO medium or other suitable cell freezing medium containing eg Hespan and PlasmaLyte A, cells are then frozen to -80°C at a rate of 1° per minute and stored in a liquid nitrogen storage tank in the gas phase. Controlled freezing as well as other methods of uncontrolled freezing at -20°C or in liquid nitrogen can be used.

In the embodiments described herein, the immune effector cells can be allogeneic immune effector cells, such as T cells or NK cells.

The T cells described herein can, for example, be engineered so that they do not express functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate cell surface expression of HLA (eg, HLA class 1 and/or HLA class II). In some aspects, HLA downregulation can be achieved by reducing or eliminating the expression of beta-2 microglobulin (B2M).

In some embodiments, T cells may lack functional TCRs and functional HLAs, such as HLA class I and/or HLA class II. Modified T cells lacking functional TCR and/or HLA expression can be obtained by any suitable means, including knockout of one or more TCR or HLA subunits or blocking gene expression. For example, T cells can be blocked using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs). Gene expression of TCR and/or HLA.

In some embodiments, an allogeneic cell can be a cell that does not express an inhibitory molecule or expresses an inhibitory molecule at low levels, eg, a cell engineered by any of the methods described herein. For example, a cell can be a cell that does not express, or at low levels, an inhibitory molecule that, for example, reduces the ability of a chimeric protein expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, 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, MHC class II, Gal9, adenosine and TGFR beta. Inhibition of inhibitory molecules, such as inhibition of DNA, RNA, or protein content, maximizes CAR-expressing cellular efficacy. In embodiments, inhibitory nucleic acids (eg, inhibitory nucleic acids, eg, dsRNA, eg, siRNA or shRNA), clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like Effector nucleases (TALENs) or zinc finger endonucleases (ZFNs).

T細胞通常可以如以下中所描述之方法活化及擴增：例如美國專利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 US Patent Application Publication No. 20060121005.

In general, T cells of the present invention can be expanded by contacting a surface to which an agent that stimulates signals associated with the CD3/TCR complex and ligands that stimulate co-stimulatory molecules on the surface of the T cells are attached. In particular, T cell populations can be stimulated as described herein, such as by interaction with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by interaction with a protein kinase C activator (eg, moss inhibitor). element) and calcium ionophore. To co-stimulate helper molecules on the surface of T cells, ligands that bind the helper molecules are used. For example, a T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. In order to stimulate the proliferation of CD4+ T cells or CD8+ T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), which can be used by other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975- 3977, 1998; Haanen et al, J. Exp. Med. 190(9):13191328, 1999; Garland et al, J. Immunol Meth. 227(1-2):53-63, 1999).

In some embodiments, amplification can be performed using flasks or vessels or gas permeable vessels known to those skilled in the art and can be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days , about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, or 13 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 14 days.

In certain embodiments, expansion can be performed using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulators can include, for example, anti-CD3 antibodies, such as about 30 ng/ml OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, N.J., or Miltenyi Biotech, Auburn, Calif. ) or UHCT-1 (available from BioLegend, San Diego, Calif., USA). Chimeric protein cells can be developed by including one or more antigens of cancer (including antigenic portions thereof such as epitopes) in the presence of T cell growth factors such as 300 IU/mL IL-2 or IL-15, as appropriate. Amplified in vitro, the one or more antigens are optionally expressed from a carrier such as a human leukocyte antigen A2 (HLA-A2) binding peptide, eg, 0.3.mu.M MART-1:26-35(27L) or gp100: 209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2 and VEGFR2 or antigenic portions thereof. Chimeric protein cells can also be rapidly expanded by restimulation with the same antigen(s) pulsed to the cancer on the antigen presenting cells expressing HLA-A2. Alternatively, the chimeric protein cells can be further stimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, stimulation occurs as part of amplification. In some embodiments, expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In certain embodiments, the cell culture medium comprises IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL , about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL, or between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 Between IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 and 9000 IU/mL of IL-2.

In certain embodiments, the cell culture medium comprises the OKT3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL , about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 μg/mL, or between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between Between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, or between 50 ng/mL and 100 ng OKT3 antibody between /mL.

In certain embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is employed as the combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during expansion. In some embodiments, a combination of IL-2, IL-15, and IL-21 is employed as the combination during expansion. In some embodiments, IL-2, IL-15, and IL-21 and any combination thereof may be included.

In certain embodiments, expansion can be performed in supplemented cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells.

In certain embodiments, the expansion medium comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15 , about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15 15, or about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15, or about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15, or about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 200 IU/mL of IL-15, or about 180 IU/mL of IL-15.

In some embodiments, the expansion medium comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21, IL-21 at about 5 IU/mL, IL-21 at about 4 IU/mL, IL-21 at about 3 IU/mL, IL-21 at about 2 IU/mL, IL-21 at about 1 IU/mL, or about 0.5 IU/mL of IL-21, or about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21 -21, or about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 5 IU/mL mL of IL-21 to about 1 IU/mL of IL-21, or about 2 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21 or about 0.5 IU/mL of IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In an embodiment, the ratio of chimeric protein cells to PBMC and/or antigen presenting cells in the expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, approximately 1:200, approximately 1:225, approximately 1:250, approximately 1:275, approximately 1:300, approximately 1:325, approximately 1:350, approximately 1:375, approximately 1:400 or approximately 1:500, or between 1:50 and 1:300, or between 1:100 and 1:200.

In certain aspects, the primary and costimulatory signals for T cells can be provided by different protocols. For example, the reagents that provide each signal can be in solution or coupled to a surface. When coupled to surfaces, the reagents can be coupled to the same surface (ie, in the "cis" form) or to a separate surface (ie, in the "trans" form). Alternatively, one reagent can be coupled to the surface and the other reagent in solution. In one aspect, the agent providing the costimulatory signal is bound to the cell surface and the agent providing the primary activation signal is in solution or coupled to the surface. In some aspects, the two reagents can be in solution. In one aspect, the agents can be in soluble form and subsequently cross-linked to a surface (such as cells expressing Fc receptors, or antibodies, or other binding agents that will bind to the agents). In this regard, see, eg, US Patent Application Publication Nos. 20040101519 and 20060034810, artificial antigen presenting cells (aAPCs), which are contemplated for use in the present invention to activate and expand T cells.

In one aspect, the two reagents are immobilized on beads, either on the same bead, ie, "cis"; or on separate beads, ie, "trans." For example, the reagent providing the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof and the reagent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and the two reagents are co-immobilized on the same bead with equivalent molecular weights . In one aspect, a 1:1 ratio of each antibody bound to beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that increased T cell expansion is observed as compared to that observed using a 1:1 ratio. In one particular aspect, an about 1-fold to about 3-fold increase is observed compared to the amplification observed using a 1:1 ratio. In one aspect, the ratio of CD3:CD28 antibody bound to the beads is in the range of 100:1 to 1:100 and all integer values in between. In one aspect of the invention, more anti-CD28 antibody is bound to the particle than anti-CD3 antibody, ie, the CD3:CD28 ratio is less than one. In certain aspects of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In a specific aspect, a 1:100 ratio of antibody CD3:CD28 bound to beads is used. In one aspect, a 1:75 ratio of antibody CD3:CD28 bound to beads is used. In another aspect, a 1:50 ratio of CD3:CD28 antibody bound to beads is used. In one aspect, a 1:30 ratio of CD3:CD28 antibody bound to the beads is used. In a preferred aspect, a 1:10 ratio of antibody CD3:CD28 bound to beads is used. In one aspect, a 1:3 ratio of CD3:CD28 antibody bound to beads is used. In yet another aspect, a 3:1 ratio of bead-bound antibody CD3:CD28 is used.

T cells or other target cells can be stimulated using particle to cell ratios of 1:500 to 500:1 and any integer value in between. As can be readily understood by those of ordinary skill, the ratio of particles to cells can be determined relative to the particle size of the target cells. For example, small beads can bind only a few cells, while larger beads can bind many cells. In certain aspects, the ratio of cells to particles is in the range of 1:100 to 100:1 and any integer value in between, and in other aspects, including 1:9 to 9:1 and any integer value in between The ratio can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupled particles to T cells resulting in T cell stimulation can vary as indicated above, however, some preferred 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, a preferred ratio is at least 1:1 particles/T cells. In one aspect, a particle to cell ratio of 1:1 or less is used. In a particular aspect, the preferred particle:cell ratio is 1:5. In other aspects, the ratio of particles to cells may vary depending on the number of days of stimulation. For example, in one aspect, the ratio of particles to cells is 1:1 to 10:1 on the first day, and additional particles are added to cells every day or every other day thereafter for up to 10 days, resulting in a final ratio of 1 :1 to 1:10 (based on cell counts on the day of addition). In one particular aspect, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis until a final ratio of 1:1 on day one, and 1:5 on days three and five of stimulation. In one aspect, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis up to a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. Those skilled in the art will appreciate that various other ratios may be suitable for the present invention. In particular, the ratio will vary depending on particle size and cell size and type. In one aspect, the most typical ratios used on the first day are about 1:1, 2:1, and 3:1.

In other aspects of the invention, cells, such as T cells, are combined with reagent-coated beads, the beads and cells are then separated, and the cells are then cultured. In an alternative aspect, the reagent-coated beads and cells are not separated but cultured together prior to culturing. In another aspect, beads and cells are first focused by applying a force, such as a magnetic force, resulting in increased engagement of cell surface markers, which in turn induces cell stimulation.

Viral and non-viral based genetic engineering tools can be used to generate chimeric protein cells, including but not limited to T cells, NK cells that result in permanent or transient expression of therapeutic genes. Retroviral-based gene delivery is a mature and well-characterized technology that has been used to permanently integrate CARs into the host cell genome (Scholler J., eg, Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells). 2012;4:132ra53; Rosenberg S.A. et al., Gene transfer into humans - immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 1990 ;323:570-578)

Non-viral DNA transfection methods can also be used. For example, Singh et al. describe the use of a Sleeping Beauty (SB) transposon system developed for engineering CAR T cells (Singh H. et al., Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res. 2008;68:2961-2971) and used in clinical trials (see eg ClinicalTrials.gov: NCT00968760 and NCT01653717). The same technique is suitable for engineering chimeric protein cells.

Transgenes have been delivered using a variety of SB enzymes. Mátés describes a highly active transposase (SB100X) that is approximately 100-fold more efficient when compared to first generation transposases. SB100X supports 35 to 50% stable gene transfer of human CD34(+) cells enriched in hematopoietic stem or precursor cells (Mátés L. et al., Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 2009;41:753-761) and multiple transgenes can be derived from polycistronic monomers (eg Thokala R. et al., Redirecting specificity of T cells using the Sleeping Beauty system to express chimeric antigen receptors by mix- and-matching of VL and VH domains targeting CD123+ tumors. PLoS ONE. 2016;11:e0159477) or multiple plastids (e.g., Hurton L.V. et al., Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc. Natl. Acad. Sci. USA. 2016;113:E7788-E7797) delivery. Such systems are used with the chimeric proteins of the present invention.

Morita et al. describe a piggyBac transposon system for integrating larger transgenes (Morita D. et al., Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol. Ther. Methods Clin. Dev. 2017 ;8:131-140). Nakazawa et al. describe the use of this system to generate EBV-specific cytotoxic T cells expressing HER2-specific chimeric antigen receptors (Nakazawa Y et al., PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther. 2011;19:2133-2143). Manuri et al. used this system to generate CD-19-specific T cells (Manuri P.V.R. et al., piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum. Gene Ther. 2010;21: 427-437).

Transposon technology is easy and economical. A potential disadvantage is that currently employed longer expansion protocols may result in T cell differentiation, diminished activity, and poor survival of infused cells. Monjezi et al. describe the development of minicircle vectors that minimize these difficulties via higher efficiency integration (Monjezi R. et al., Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia. 2017; 31:186-194). These transposon technologies can be used in the chimeric proteins of the present invention.

The present invention also relates to a pharmaceutical composition comprising the carrier or chimeric protein-expressing cell of the present invention, a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more other pharmaceutically active polypeptides and / or compound.

In some embodiments, there is provided a pharmaceutical composition comprising the chimeric protein described above and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising a nucleic acid encoding a chimeric protein according to any of the above-described embodiments and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising effector cells expressing the chimeric protein described above and a pharmaceutically acceptable carrier. Such formulations may, for example, be in a form suitable for intravenous infusion.

As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is biologically or otherwise not adversely affected, eg, can be incorporated into a pharmaceutical composition administered to a patient without causing any significant adverse biological effects or interacting in a detrimental manner with any other component of the composition containing it. The pharmaceutically acceptable carrier or excipient preferably meets the necessary criteria for toxicology and manufacturing testing and/or includes the Inactive Ingredient Guidelines (Inactive Ingredient) established by the U.S. Food and Drug Administration. Guide).

One aspect of the present invention provides populations of modified T cells expressing recombinant chimeric proteins. Suitable populations can be generated by the methods described above.

Populations of modified T cells can be used as pharmaceutical agents. For example, populations of modified T cells as described herein can be used in cancer immunotherapy therapy, such as adoptive T cell therapy.

Other aspects of the invention provide the use of a population of modified T cells as described herein for the manufacture of a medicament for treating cancer, a population of modified T cells as described herein for treating cancer, and methods of treating cancer Administration of a population of modified T cells as described herein to an individual in need thereof can be included.

A population of modified T cells can be autologous, ie, the modified T cells were originally obtained from the same individual to which they were subsequently administered (ie, the donor and recipient individuals are the same). A population of modified T cells suitable for administration to an individual can be generated by a method comprising: providing a naive population of T cells obtained from the individual; modifying the T cells to express cAMP PDE or a fragment thereof and specifically bind to the subject antigen receptors of cancer cells; and cultured modified T cells.

A population of modified T cells can be allogeneic, ie, the modified T cells are initially obtained from different individuals to the subjects to which they are subsequently administered (ie, the donor and recipient subjects are different). Donor and recipient individuals may be HLA matched to avoid GVHD and other undesirable immune effects. Populations of modified T cells suitable for administration to recipient individuals can be generated by methods comprising: providing an initial population of T cells obtained from a donor subject; modifying T cells to exhibit specific binding to the recipient subject chimeric cells in cancer cells; and cultured modified T cells.

Following administration of the modified T cells, the recipient individual can exhibit a T cell-mediated immune response against cancer cells in the recipient individual. This can have a beneficial effect on the condition of cancer in an individual.

Cancer conditions can be characterized by abnormal proliferation of malignant cancer cells and can include leukemias, such as AML, CML, ALL, and CLL; lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma; and Solid cancers such as sarcoma, skin cancer, melanoma, bladder, brain, breast, uterus, ovary, prostate, lung, colorectal, cervix, liver, head and neck, esophagus, pancreas , kidney cancer, adrenal cancer, gastric cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, brain cancer and cancer of unknown primary (CUP).

Cancer cells in an individual can be immunologically distinct from normal somatic cells in a subject (ie, cancerous tumors can be immunogenic). For example, cancer cells may be able to induce a systemic immune response in an individual against one or more antigens displayed by the cancer cells. Tumor antigens that elicit an immune response may be specific to cancer cells or may be shared by one or more normal cells in an individual.

Individuals suitable for treatment as described above may be mammals, such as rodents (eg, guinea pigs, hamsters, rats, mice), murine (eg, mice), canines (eg, dogs), felines (eg, cats) ), equine (eg, horse), primate, simian (eg, monkey or great ape), monkey (eg, marmoset, baboon), ape (eg, gorilla, chimpanzee, orangutan, gibbon), or human.

In a preferred embodiment, the individual is a human. In other preferred embodiments, non-human mammals may be employed, particularly mammals conventionally used as models for demonstrating therapeutic efficacy in humans (eg, murine, primate, porcine, canine, or rabbit animals).

The term "therapeutically effective amount" refers to an amount of a chimeric protein or a composition comprising a chimeric protein as disclosed herein that is effective to "treat" a disease or disorder in an individual. In the case of cancer, a therapeutically effective amount of a chimeric protein or a composition comprising a chimeric protein as disclosed herein can reduce the number of cancer cells; reduce tumor size or weight; preferably stop) infiltration of cancer cells into surrounding organs; inhibit (ie, to some extent slow and preferably stop) tumor metastasis; to some extent inhibit tumor growth; related symptoms. To the extent that a chimeric protein or a composition comprising a chimeric protein as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, a therapeutically effective amount is an amount that improves progression-free survival in a patient. In the case of infectious diseases such as viral infections, a therapeutically effective amount of a chimeric protein or a composition comprising a chimeric protein as disclosed herein can reduce the number of cells infected by a pathogen; reduce the production of pathogen-derived antigens or release; inhibit (ie, to some extent slow and preferably stop) the spread of pathogens into uninfected cells; and/or alleviate to some extent one or more symptoms associated with infection. In some embodiments, a therapeutically effective amount is an amount that prolongs patient survival.

Chimeric protein-expressing cells, including T and NK cells, for use in the methods of the invention can be produced ex vivo from the patient's own peripheral blood (autologous), or in peripheral blood from a donor (allogeneic) or unconnected donor. produced in the context of hematopoietic stem cell transplantation in peripheral blood of the body (allogeneic). Alternatively, T cells or NK cells can be derived from in vivo differentiation of inducible precursor cells or embryonic precursor cells to T cells or NK cells. In these cases, by introducing DNA or RNA encoding the chimeric protein and optionally the CAR and/or TCR, by one of many means including transduction with viral vectors, transfection with DNA or RNA T cells expressing the chimeric protein and optionally CAR and/or TCR are generated.

T or NK cells expressing the chimeric proteins of the present invention, and optionally TCR and/or CAR, can be used to treat hematological cancers or solid tumors.

Methods for treating disease relate to the therapeutic use of the vectors or cells of the invention, including T or NK cells. In this regard, the vector or T or NK cells can be administered to a subject with an existing disease or condition in order to alleviate, reduce or ameliorate at least one disease-related symptom and/or slow, reduce or block disease progression. The methods of the present invention can cause or promote T cell mediated killing of cancer cells. The vector or T or NK cells according to the invention can be administered to a patient together with one or more additional therapeutic agents. One or more additional therapeutic agents can be co-administered to the patient. "Co-administration" means administering one or more additional therapeutic agents and a vector or T or NK cell of the invention sufficiently close in time so that the vector or T or NK cell can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the vector or cell can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the vector or cells and one or more additional therapeutic agents can be administered simultaneously. One potentially useful co-administered therapeutic agent is IL-2, as it is currently used in existing cell therapy to enhance the activity of administered cells. However, IL-2 therapy is associated with toxicity and tolerability issues.

As mentioned, for administration to a patient, the chimeric protein effector cells can be allogeneic or autologous to the patient. In certain embodiments, the allogeneic cells are further genetically modified, such as by gene editing, to minimize or prevent GVHD and/or patient immune responses to the chimeric protein cells.

Chimeric protein effector cells are used to treat cancers and neoplastic diseases associated with target antigens. Cancers and neoplastic diseases that can be treated using any of the methods described herein include tumors that are not vascularized or not substantially vascularized, as well as vascularized tumors. Cancers may comprise non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may comprise solid tumors. The types of cancers to be treated with the chimeric protein effector cells of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and, for example, sarcomas, carcinomas Malignant diseases such as melanoma. Adult Oncology/Cancer and Pediatric Oncology/Cancer are also included.

Blood cancers are cancers of the blood or bone marrow. Examples of hematological (or bloody) cancers include leukemias, including acute leukemias such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic, monocytic erythrocytic leukemia), chronic leukemia (such as chronic myelocytic (myeloid) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease disease), non-Hodgkin's lymphoma (refractory and advanced forms), multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia, and myelodysplasia.

Solid tumors are abnormal tissue masses that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell type that forms them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma , rhabdomyosarcoma, colon cancer, gastric cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid carcinoma (e.g. , medullary thyroid cancer and papillary thyroid cancer), pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer (eg, cervical carcinoma and preinvasive cervical dysplasia), colorectal, anal, anal or anorectal, vaginal, vulvar (eg. , squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma), penile carcinoma, oropharyngeal carcinoma, esophageal carcinoma, head carcinoma (eg, squamous cell carcinoma), neck cancer (eg, squamous cell carcinoma), testicular carcinoma Carcinomas (eg, sperm cell carcinoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, Leydig cell tumor, fibroma, fibroadenoma, adenomatous tumor, and lipoma), bladder cancer, kidney cancer, melanoma, uterine cancer (eg, endometrial cancer), urothelial cancer (eg, squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureteral carcinoma, and bladder cancer), and CNS tumors (such as nerve Glioma (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma pleomorphism), astrocytoma, CNS lymphoma, blastoma, neural tube blastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

When indicating an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount" or "therapeutic amount", the precise amount to be administered with the composition of the present invention can be determined by the physician taking into account the age, weight, tumor size of the individual , the degree of infection or metastasis and the difference in the symptoms of patients (subjects). It can generally be specified that a pharmaceutical composition comprising the T cells described herein can range from 10 ⁴ to 10 ⁹ cells/kg body weight, in some cases 10 ⁵ to 10 ⁶ cells/kg body weight (including all integer values within those ranges). ) dose administered. The T-cell composition can also be administered in multiple doses of these same doses. Cells can be administered by using infusion techniques commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

The chimeric protein expressing cells described herein can be used in combination with other known agents and therapies. As used herein, "combination" administration means delivering two (or more) different treatments to the subject during the course of the subject afflicted with the disorder, eg, after the subject has been diagnosed with Two or more treatments are delivered after the disorder and until the disorder is cured or eliminated or treatment is discontinued for other reasons. In some embodiments, when delivery of the second treatment is initiated, the delivery of the first treatment is still present such that there is overlap in administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery." In other embodiments, delivery of one therapy ends before delivery of another therapy begins. In some embodiments of either case, the therapy is more effective due to the combined administration. For example, the second treatment may be seen to be more effective than the administration of the second treatment in the absence of the first treatment, eg, the same effect is seen with less second treatment, or the second treatment reduces symptoms to a greater extent , or a similar situation can be seen for the first treatment. In some embodiments, the delivery results in a reduction in symptoms, or other parameters associated with the condition, greater than those that would be observed if one treatment were delivered in the absence of another treatment. The effects of the two treatments can be partially additive, fully additive or greater than additive. The delivery can be such that the effects of the first treatment delivered are still detectable when the second treatment is delivered.

The chimeric protein expressing cells described herein and the at least one additional therapeutic agent can be administered simultaneously or sequentially in the same or separate compositions. For sequential administration, the CAR-expressing cells described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

Chimeric protein therapy and/or other therapeutic agents, procedures or modalities are administered during periods of active disease or during periods of remission or less active disease. Chimeric protein therapy can be administered before, concurrently with, post-treatment, or during remission of the disorder.

When administered in combination, the therapy and the additional agent (eg, a second or third agent), or all, may be administered in a higher, lower, or the same amount or dose than the amount or dose of each agent used independently, eg, as a monotherapy and. In certain embodiments, the amount or dose of the chimeric protein therapy, the additional agent (eg, a second or third agent), or all administered is lower than the amount or dose of each agent used independently, eg, as a monotherapy ( For example at least 20%, at least 30%, at least 40% or at least 50%). In other embodiments, the amount or dose of the chimeric protein therapy, the additional agent (eg, a second or third agent), or all, that causes the desired effect (eg, cancer treatment) is used independently of each agent, eg, in a monotherapy format to achieve The amount or dose required for the same therapeutic effect is lower (eg, at least 20% lower, at least 30% lower, at least 40% lower, or at least 50% lower).

In other aspects, the chimeric protein expressing cells described herein can be used in therapeutic regimens and surgery, chemotherapy, radiation, immunosuppressants (such as cyclosporin, azathioprine, methotrexate) (methotrexate, mycophenolate and FK506), antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibody or other antibody therapy, cytotoxins, fludarabine, cyclosporine, FK506, A combination of rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiotherapy, peptide vaccines such as those described in Izumoto et al. 2008 J Neurosurg 108:963-971.

In certain instances, the compounds of the present invention are combined with other therapeutic agents, such as other anticancer agents, antiallergic agents, antinausea agents (or antiemetic agents), pain relievers, cytoprotective agents, and combinations thereof.

In one embodiment, the chimeric protein expressing cells described herein can be used in combination with chemotherapeutic agents. Exemplary chemotherapeutic agents include anthracycline (eg, doxorubicin (eg, liposomal doxorubicin)), vinca alkaloid (eg, vinblastine) ), vincristine, vindesine, vinorelbine), alkylating agents (eg, cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (eg, alemtuzamab, gemtuzumab, rituximab, ofatumumab ofatumumab, tositumomab, brentuximab), antimetabolites (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors ( e.g., fludarabine), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin ) or bortezomib), immunomodulators (such as thalidomide or thalidomide derivatives (eg, lenalidomide)).

Common chemotherapeutic agents considered for use in combination therapy include busulfan (Myleran®), busulfan injection (Busulfex®), cladribine (Leustatin®), cyclophosphamide (Cytoxan) ® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®), lemon Daunomycin acid liposome injection (DaunoXome®), dexamethasone (dexamethasone), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), phosphoric acid Fludarabine (Fludara®), Hydroxyurea (Hydrea®), Idarubicin (Idamycin®), mitoxantrone (Novantrone®), zistuzumab, ogituzumab (Gemtuzumab Ozogamicin) (mylotarg®).

In an embodiment, typical chemotherapeutic agents contemplated for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate ( Blenoxane®), Busulfan (Myleran®), Busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytosine glycosides, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (cladribine) (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injectable (DepoCyt®), dacarbazine (DTIC-Dome®), actinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome ®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), phosphoric acid fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluoro) difluorodeoxycitidine), hydroxyurea (Hydrea®), idamycin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L -Asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (yttrium 90/MX- DTPA), pentostatin, polyproxil 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine ( Velban®), vincristine (Oncovin®) and vinorelbine (Navelbine®).

Treatment can be assessed, for example, by tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Methods of determining the efficacy of therapy can be employed, including, for example, measuring response via radiographic imaging.

TCR-incorporated antigen-indeterminate receptor (TIAAR)

Table 1 provides illustrative, non-limiting examples of components of the TCR-incorporated antigen-indeterminate receptors (TIAARs) of the invention. Table 2 shows exemplary configurations of components. Table 1 - TCR-Incorporated Antigen Indeterminate Receptor (TIAAR) Components code signal peptide Label ECD_TMD ICD Costim pIB1026 CD3D Myc CD3D N/A CD28-CD40 pIB1027 CD3E FLAG CD3E N/A CD28-CD40 pIB1028 CD3G Myc CD3G N/A CD28-CD40 pIB1029 CD3Z Myc IC CD3Z N/A CD28-CD40 pIB1030 CD8A Myc hTRDC N/A CD28-CD40 pIB1031 CD8A FLAG hTRGC1 N/A CD28-CD40 pIB1032 CD8A Myc mTRAC N/A CD28-CD40 pIB1033 CD8A FLAG mTRBC1 N/A CD28-CD40 pIB1046 CD8A x2 Myc and FLAG hTRDC_hTRGC1 N/A CD28-CD40 pIB1047 CD8A x2 Myc and FLAG mTRAC_mTRBC1 N/A CD28-CD40 pIB1048 CD3D and CD3E Myc and FLAG CD3D_CD3E N/A CD28-CD40 pIB1049 CD3G and CD3E Myc and FLAG CD3G_CD3E N/A CD28-CD40 pIB1050 CD3D and CD3E Myc and FLAG CD3D_CD3E CD3D_CD3E CD28-CD40 pIB1051 CD3D and CD3E Myc and FLAG CD3D_CD3E CD3D_CD3E N/A pIB1052 CD3D and CD3E Myc and FLAG CD3D_CD3E N/A N/A pIB1053 CD3G and CD3E Myc and FLAG CD3G_CD3E CD3G_CD3E CD28-CD40 pIB1054 CD3G and CD3E Myc and FLAG CD3G_CD3E CD3G_CD3E N/A pIB1055 CD3G and CD3E Myc and FLAG CD3G_CD3E N/A N/A pIB1056 CD3Z Myc IC CD3Z CD3Z CD28-CD40 pIB1057 CD3Z Myc IC CD3Z CD3Z N/A pIB1058 CD3Z Myc IC CD3Z N/A N/A pIB1059 CD3Z Myc IC CD3Z CD3Z (x2) CD28-CD40 pIB1060 CD3Z Myc IC CD3Z CD3Z (x2) N/A pIB1061 CD3Z Myc IC CD3Z CD3Z (x2) CD28-CD40 (swap) pIB1062 CD3Z Myc IC CD3Z CD3Z CD28-CD40 (swap) pIB1063 CD80 Myc CD80 CD80 N/A pIB1064 No signal peptide Myc IC N/A Lck N/A pIB1065 No signal peptide Myc IC N/A Lck (Y505F) N/A pIB1066 CD80 Myc CD80 Lck N/A pIB1067 CD80 Myc CD80 Lck CD28-CD40 pIB1068 CD80 Myc CD80 CD80_Lck (Y505F) N/A pIB1069 CD80 Myc CD80 CD80_Lck (Y505F) CD28-CD40 pIB1070 CD8A Myc LAT LAT N/A pIB1071 CD8A Myc LAT LAT CD28-CD40 pIB1072 CD4 Myc CD4 CD4 CD28-CD40 pIB1073 CD4 Myc CD4 CD4 CD28-CD40 pIB1074 CD8A and CD8B Myc and FLAG CD8A and CD8B CD8A and CD8B N/A pIB1075 CD8A and CD8B Myc and FLAG CD8A and CD8B CD8A and CD8B CD28-CD40 Table 2 - TCR-Incorporated Antigen Indeterminate Receptors (TIAARs) code describe pIB1026 CD3D_CD3D_CD28CD40 pIB1027 CD3E_CD3E_CD28CD40 pIB1028 CD3G_CD3G_CD28CD40 pIB1029 CD3Z_CD3Z_CD28CD40_Myc pIB1030 CD8A_hTRDC_CD28CD40 pIB1031 CD8A_hTRGC1_CD28CD40 pIB1032 CD8A_mTRAC_CD28CD40 pIB1033 CD8A_mTRBC1_CD28CD40 pIB1046 CD8A_hTRDC_CD28CD40-T2A-CD8a_hTRGC1_CD28CD40 pIB1047 CD8A_mTRAC_CD28CD40-T2A-CD8A_mTRBC1_CD28CD40 pIB1048 CD3D_CD3D_CD28CD40-T2A-CD3E_CD3E_CD28CD40 pIB1049 CD3G_CD3G_CD28CD40-T2A-CD3E_CD3E_CD28CD40 pIB1050 CD3D_CD3D_CD3D (ICD)_CD28CD40-T2A-CD3E_CD3E_CD3E (ICD)_CD28CD40 pIB1051 CD3D_CD3D_CD3D ICD -T2A-CD3E_CD3E_CD3E ICD pIB1052 CD3D_CD3D (control)-T2A-CD3E_CD3E (control) pIB1053 CD3G_CD3G_CD3G (ICD)_CD28CD40-T2A-CD3E_CD3E_CD3E (ICD)_CD28CD40 pIB1054 CD3G_CD3G_CD3G ICD -T2A-CD3E_CD3E_CD3E ICD pIB1055 CD3G_CD3G (control)-T2A-CD3E_CD3E (control) pIB1056 CD3z _CD3z_CD3z ICD _CD28CD40_Myc pIB1057 CD3z _CD3z_CD3z ICD_Myc pIB1058 CD3z_CD3z (control) pIB1059 CD3Z_CD3Z_CD3Z ICD (repeat CD3z intracellular domain-6 ITAMs) _CD28CD40_Myc pIB1060 CD3Z_CD3Z_CD3Z ICD (repeat CD3z intracellular domain-6 ITAMs)_Myc pIB1061 CD3Z_CD3Z_CD28CD40_CD3Z (6 ITAMs)_Myc pIB1062 CD3Z_CD3Z_CD28CD40_CD3Z ICD_Myc pIB1063 CD80 (control) pIB1064 Lck (control) pIB1065 Lck (Y505F) (control) pIB1066 CD80_Lck pIB1067 CD80_Lck_CD28CD40 pIB1068 CD80_Lck (Y505F) pIB1069 CD80_Lck (Y505F)_CD28CD40 pIB1070 LAT (control) pIB1071 LAT_C28CD40 pIB1072 CD4 control pIB1073 CD4_CD28_CD40 pIB1074 CD8 control pIB1075 CD8_CD28_CD40

constitutive costimulatory protein

Table 3 provides illustrative, non-limiting examples of components of the constitutive costimulatory proteins of the present invention. Table 4 shows exemplary configurations of components. Table 3 - Constitutive Stimulating Antigen Undetermined Receptor (C-SAAR) Components code signal peptide Label ECD_TMD Costim pIB1076 CD8A Myc LZ (cFos)_EGFR CD28-CD40 pIB1077 CD8A Myc LZ (cFos)_CD28 CD28-CD40 pIB1078 CD8A Myc LZ (cJun)_EGFR CD28-CD40 pIB1079 CD8A Myc LZ (cJun)_CD28 CD28-CD40 pIB1080 CD8A Myc LZ (c/EBP)_EGFR CD28-CD40 pIB1081 CD8A Myc LZ (c/EBP)_CD28 CD28-CD40 pIB1103 GpA Myc GpA ECD_TMD CD28-CD40 pIB1104 GpA Myc GpA TMD CD28-CD40 pIB1105 EPOR Myc EPOR ECD_TMD CD28-CD40 pIB1106 EPOR Myc EPORTM CD28-CD40 pIB1107 TPOR Myc TPOR ECD_TMD CD28-CD40 pIB1108 TPOR Myc TPOR TMD CD28-CD40 pIB1109 TPOR Myc TPOR ECD_TMD (S505N) CD28-CD40 pIB1110 TPOR Myc TPOR TMD (S505N) CD28-CD40 pIB1111 TPOR Myc TPOR ECD_TMD (W515K) CD28-CD40 pIB1112 TPOR Myc TPOR TMD (W515K) CD28-CD40 pIB1113 TPOR Myc TPOR ECD_TMD (H499L) CD28-CD40 pIB1114 TPOR Myc TPOR TMD (H499L) CD28-CD40 pIB1115 TPOR Myc TPOR ECD_TMD (S505N-W515K) CD28-CD40 pIB1116 TPOR Myc TPOR TMD (S505N-W515K) CD28-CD40 pIB1117 TPOR Myc TPOR ECD_TMD (H499Y-S505N) CD28-CD40 pIB1118 TPOR Myc TPOR TMD (H499Y-S505N) CD28-CD40 pIB1119 TPOR Myc TPOR ECD_TMD (L498W-H499C) CD28-CD40 pIB1120 TPOR Myc TPOR TMD (L498W-H499C) CD28-CD40 pIB1025 CD8a Myc CD28 CD28-CD40 pIB1179 CD8a N/A IgG1+CD28TM CD28-CD40 pIB1180 CD8a N/A IgG1mut+CD28TM CD28-CD40 pIB1181 CD8a N/A IgG2+CD28TM CD28-CD40 pIB1182 CD8a N/A IgG3+CD28TM CD28-CD40 pIB1183 CD8a N/A IgG4+CD28TM CD28-CD40 pIB1184 CD8a N/A IgG4mut+CD28TM CD28-CD40 pIB1185 CD8a N/A IgG1 + CD28 STEM/TM CD28-CD40 pIB1186 CD8a N/A IgG1mut + CD28 STEM/TM CD28-CD40 pIB1187 CD8a N/A IgG2 + CD28 STEM/TM CD28-CD40 pIB1188 CD8a N/A IgG3 + CD28 STEM/TM CD28-CD40 pIB1189 CD8a N/A IgG4 + CD28 STEM/TM CD28-CD40 pIB1190 CD8a N/A IgG4mut + CD28 STEM/TM CD28-CD40 Table 4 - C-SAAR proteins code describe pIB1076 LZ (cFos)-EGFRTM/JMD-CD28-CD40 pIB1077 LZ (cFos)-CD28TM-CD28-CD40 pIB1078 LZ (cJun)-EGFRTM/JMD-CD28-CD40 pIB1079 LZ (cJun)-CD28TM-CD28-CD40 pIB1080 LZ (c/EBP)-EGFRTM/JMD-CD28-CD40 pIB1081 LZ(c/EBP)-CD28TM-CD28-CD40 pIB1103 GpA ECD-TMD-CD28-CD40 pIB1104 GpA TMD-CD28-CD40 pIB1105 EpoR ECD-TMD-CD28-CD40 pIB1106 EpoR TMD-CD28-CD40 pIB1107 TPO ECD- TPO (WT) TMD-CD28-CD40 pIB1108 TPO (WT) TMD-CD28-CD40 pIB1109 TPO ECD- TPO (S505N) TMD-CD28-CD40 pIB1110 TPO (S505N) TMD-CD28-CD40 pIB1111 TPO ECD- TPO (W515K) TMD-CD28-CD40 pIB1112 TPO (W515K) TMD-CD28-CD40 pIB1113 TPO ECD- TPO (H499L) TMD-CD28-CD40 pIB1114 TPO (H499L) TMD-CD28-CD40 pIB1115 TPO ECD- TPO (S505N-W515K) TMD-CD28-CD40 pIB1116 TPO (S505N-W515K) TMD-CD28-CD40 pIB1117 TPO ECD- TPO (H499Y-S505N) TMD-CD28-CD40 pIB1118 TPO (H499Y-S505N) TMD-CD28-CD40 pIB1119 TPO ECD-TPO (L498W-H499C) TMD-CD28-CD40 pIB1120 TPO (L498W-H499C) TMD-CD28-CD40 pIB1025 CD28TM_CD28_CD40 pIB1179 IgG1(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1180 IgG1(CH2CH3, mutant)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1181 IgG2(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1182 IgG3(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1183 IgG4(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1184 IgG4(CH2CH3, mutant)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1185 IgG1(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1186 IgG1(CH2CH3, mutant)-CD28(STEM+TM)-CD28(CoStim)-CD40(CoStim) pIB1187 IgG2(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1188 IgG3(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1189 IgG4(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1190 IgG4(CH2CH3, mutant)-CD28(STEM+TM)-CD28(CoStim)-CD40(CoStim)

inducible costimulatory receptor

Table 5 provides illustrative non-limiting examples of inducible costimulatory receptors of the present invention. Table 6 shows exemplary configurations of components. Table 5 - Inducible costimulatory protein components code signal peptide Label ECD_TMD ICD Costim pIB1082 EGFR Myc EGFR N/A CD28-CD40 pIB1083 EGFR Myc EGFR (Domain IV) N/A CD28-CD40 pIB1084 EGFR Myc EGFR (623-668) N/A CD28-CD40 pIB1085 Her2 Myc Her2 N/A CD28-CD40 pIB1086 Her2 Myc Her2 (V659E) N/A CD28-CD40 pIB1087 Her2 Myc Her2 (V660D) N/A CD28-CD40 pIB1088 Her2 Myc Her2 (V660R) N/A CD28-CD40 pIB1089 Her2 Myc Her2 Domain IV_TMD N/A CD28-CD40 pIB1090 Her2 Myc Her2 Domain IV_TMD (V659E) N/A CD28-CD40 pIB1091 Her2 Myc Her2 Domain IV_TMD (G660D) N/A CD28-CD40 pIB1092 Her2 Myc Her2 Domain IV_TMD (G660R) N/A CD28-CD40 pIB1093 Her2 Myc Her2 TMD N/A CD28-CD40 pIB1094 Her2 Myc Her2 TMD (V659E) N/A CD28-CD40 pIB1095 Her2 Myc Her2 TMD (G660D) N/A CD28-CD40 pIB1096 Her2 Myc Her2 TMD (G660R) N/A CD28-CD40 pIB1097 CD8A Myc A30514 VH_VL N/A CD28-CD40 pIB1098 CD8A Myc A30514 VL_VH N/A CD28-CD40 pIB1099 CD8A Myc A30523 VH_VL N/A CD28-CD40 pIB1100 CD8A Myc A30523 VL_VH N/A CD28-CD40 pIB1101 CD8A Myc A30633 VH_VL N/A CD28-CD40 pIB1102 CD8A Myc A30633 VL_VH N/A CD28-CD40 Table 6 - Inducible costimulatory proteins code GOI description pIB1082 WT EGFR ECD-EGFRTM/JMD-CD28-CD40 pIB1083 Domain IV - EGFRTM/JMD-CD28-CD40 pIB1084 EGFRTM/JMD-CD28-CD40 (control) pIB1085 Her 2 (Domains 1 to IV)-TMD/JMD-CD28-CD40 pIB1086 Her 2 (Domains 1 to IV)-TMD (V659E)/JMD-CD28-CD40 pIB1087 Her 2 (Domains 1 to IV)-TMD (G660D)/JMD-CD28-CD40 pIB1088 Her 2 (Domains 1 to IV)-TMD (G660R)/JMD-CD28-CD40 pIB1089 Her 2 (Domain IV)-TMD/JMD-CD28-CD40 pIB1090 Her 2 (Domain IV)-TMD (V659E)/JMD-CD28-CD40 pIB1091 Her 2 (Domain IV)-TMD (G660D)/JMD-CD28-CD40 pIB1092 Her 2 (Domain IV)-TMD (G660R)/JMD-CD28-CD40 pIB1093 Her 2 TMD/JMD-CD28-CD40 pIB1094 Her 2 TMD (V659E)/JMD-CD28-CD40 pIB1095 Her 2 TMD (G660D)/JMD-CD28-CD40 pIB1096 Her 2 TMD (G660R)/JMD-CD28-CD40 pIB1097 Anti-ID1 VH-VL (A30514-Pembrolizumab)- CD28TMD CD28-CD40 pIB1098 Anti-ID1 VL-VH (A30514-Pembrolizumab)- CD28TMD CD28-CD40 pIB1099 Anti-ID2 Vh-VL (A30523-Pembrolizumab)- CD28TMD CD28-CD40 pIB1100 Anti-ID2 VL-Vh (A30523-Pembrolizumab)- CD28TMD CD28-CD40 pIB1101 Anti-ID3 Vh-VL (A30633-Pembrolizumab)- CD28TMD CD28-CD40 pIB1102 Anti-ID3 VL-VH (A30633-Pembrolizumab)- CD28TMD CD28-CD40

The following sequences in the table below include complete components and are non-limiting. Components may include a signal peptide (SP), a TCR clustering domain (CD) and/or a signaling domain (SD). It should be understood that while certain proteins may contain an N-terminal signal peptide when expressed, those signal peptides are cleaved and may be cleaved imprecisely when the protein is expressed, and the resulting protein from which the signal peptide is removed is contained before the N-terminal amino acid. Binding domains with variations of up to about five amino acids at positions.

Table: Examples of TCR co-stimulatory constructs SEQ ID NO: 1    Components: SP CD3D_ sequence: MEHSTFLSGL VLATLLSQVS P SEQ ID NO: 2    Components: CD CD3D_ sequence: FKIPIEELED RVFVNCNTSI TWVEGTVGTL LSDITRLDLG KRILDPRGIY RCNGTDIYKD KESTVQVHYR MCQSCVELDP ATVAGIIVTD VIATLLLALG VFCFA SEQ ID NO: 3    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 4    full length: CD3D_CD3D_CD28CD40 sequence: MEHSTFLSGL VLATLLSQVS PFKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARSKR SRLLHSDYMN MTPRRPGPTR KHYQPVTYAPPR DFAAYRSKKV AKKPTNKAPH PKLEPQEINF PDDLQSNEDQPVQETERQ SEQ ID NO: 5    Components: SP CD3E_ sequence: MQSGTHWRVL GLCLLSVGVW GQ SEQ ID NO: 6    Components: CD CD3E_ sequence: DGNEEMGGIT QTPYKVSISG TTVILTCPQY PGSEILWQHN DKNIGGDEDD KNIGSDEDHL SLKEFSELEQ SGYYVCYPRG SKPEDANFYL YLRARVCENC MEMDVMSVAT IVIVDICITG GLLLLVYYWS SEQ ID NO: 7    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 8    full length: CD3E_CD3E_CD28CD40 sequence: MQSGTHWRVL GLCLLSVGVW GQDGNEEMGG ITQTPYKVSI SGTTVILTCPQ YPGSEILWQH NDKNIGGDED DKNIGSDEDH LSLKEFSELE QSGYYVCYPRG SKPEDANFYL YLRARVCENC MEMDVMSVAT IVIVDICITG GLLLLVYYWSR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQERQ SEQ ID NO: 9    Components: SP CD3G_ sequence: MEQGKGLAVL ILAIILLQGT LA SEQ ID NO: 10    Components: CD CD3G_ sequence: QSIKGNHLVK VYDYQEDGSV LLTCDAEAKN ITWFKDGKMI GFLTEDKKKW NLGSNAKDPR GMYQCKGSQN KSKPLQVYYR MCQNCIELNA ATISGFLFAE IVSIFVLAVG VYFIA SEQ ID NO: 11    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 12    full length: CD3G_CD3G_CD28CD40 sequence: MEQGKGLAVL ILAIILLQGT LAQSIKGNHL VKVYDYQEDG SVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 13    Components: SP CD3Z_ sequence: MKWKALFTAA ILQAQLPITE A SEQ ID NO: 14    Components: CD CD3Z_ sequence: QSFGLLDPKL CYLLDGILFI YGVILTALFL SEQ ID NO: 15    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 16    full length: CD3Z_CD3Z_CD28CD40 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 17    Components: SP CD8A_ sequence: MALPVTALLL PLALLLHAAR P SEQ ID NO: 18    Components: CD TRDC_ sequence: SQPHTKPSVF VMKNGTNVAC LVKEFYPKDI RINLVSSKKI TEFDPAIVIS PSGKYNAVKL GKYEDSNSVT CSVQHDNKTV HSTDFEVKTD STDHVKPKET ENTKQPSKSC HKPKAIVHTE KVNMMSLTVL GLRMLFAKTV AVNFLLTAKL FFL SEQ ID NO: 19    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 20    full length: CD8A_TRDC_CD28CD40 sequence: MALPVTALLL PLALLLHAAR PSQPHTKPSV FVMKNGTNVA CLVKEFYPKD IRINLVSSKK ITEFDPAIVI SPSGKYNAVK LGKYEDSNSV TCSVQHDNKT VHSTDFEVKT DSTDHVKPKE TENTKQPSKS CHKPKAIVHT EKVNMMSLTV LGLRMLFAKT VAVNFLLTAK LFFLRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 21    Components: SP CD8A_ sequence: MALPVTALLL PLALLLHAAR P SEQ ID NO: 22    Components: CD TRGC1_ sequence: DKQLDADVSP KPTIFLPPSIA ETKLQKAGTY LCLLEKFFPD VIKIHWQEKK SNTILGSQEG NTMKTNDTYM KFSWLTVPEK SLDKEHRCIV RHENNKNGVD QEIIFPPIKT DVITMDPKDN CSKDANDTLL LQLTNTSAYY MYLLLLLKSV VYFAIITCCL L SEQ ID NO: 23    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 24    full length: CD8A_TRGC1_CD28CD40 sequence: MALPVTALLL PLALLLHAAR PDKQLDADVS PKPTIFLPSI AETKLQKAGT YLCLLEKFFP DVIKIHWQEK KSNTILGSQE GNTMKTNDTY MKFSWLTVPE KSLDKEHRCI VRHENNKNGV DQEIIFPPIK TDVITMDPKD NCSKDANDTL LLQLTNTSAY YMYLLLLLKS VVYFAIITCC LLRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 25    Components: SP CD8A_ sequence: MALPVTALLL PLALLLHAAR P SEQ ID NO: 26    Components: CD TRAC_ sequence: IQNPEPAVYQ LKDPRSQDST LCLFTDFDSQ INVPKTMESG TFITDKCVLD MKAMDSKSNG AIAWSNQTSF TCQDIFKETN ATYPSSDVPC DATLTEKSFE TDMNLNFQNL LVIVLRILLL KVAGFNLLMT LRLWSS SEQ ID NO: 27    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPH PKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 28    full length: CD8A_TRAC_CD28CD40 sequence: MALPVTALLL PLALLLHAAR PIQNPEPAVY QLKDPRSQDS TLCLFTDFDS QINVPKTMES GTFITDKCVL DMKAMDSKSN GAIAWSNQTS FTCQDIFKET NATYPSSDVP CDATLTEKSF ETDMNLNFQN LLVIVLRILL LKVAGFNLLM TLRLWSSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 29    Components: SP CD8A_ sequence: MALPVTALLL PLALLLHAAR P SEQ ID NO: 30    Components: CD TRBC1_ sequence: VLTPPKVSLF EPSKAEIANK QKATLVCLAR GFFPDHVELS WWVNGKEVHS GVCTDPQAYK ESNYSYCLSS RLRVSATFWH NPRNHFRCQV QFHGLSEEDK WPEGSPKPVT QNISAEAWGR ADCGITSASY QQGVLSATIL YEILLGKATL YAVLVSTLVV MAMVKRKNS SEQ ID NO: 31    Components: SD CD28CD40 sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 32    full length: CD8A_TRBC1_CD28CD40 sequence: MALPVTALLL PLALLLHAAR PVLTPPKVSL FEPSKAEIAN KQKATLVCLA RGFFPDHVEL SWWVNGKEVH SGVCTDPQAY KESNYSYCLS SRLRVSATFW HNPRNHFRCQ VQFHGLSEED KWPEGSPKPV TQNISAEAWG RADCGITSAS YQQGVLSATI LYEILLGKAT LYAVLVSTLV VMAMVKRKNS RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 33    Vector pure line: pIB1001 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGA EVKKPGASVK VSCKASGYTF TSYWMNWVRQ APGQGLEWMG RIDPYDSETH YAQKLQGRVT MTTDTSTSTA YMELRSLRSD DTAVYYCARG GYDFDVGTLY WFFDVWGQGT TVTVSSGGGG SGGGGSGGGG SDIQMTQSPS SLSASVGDRV TITCRASENI YSYLAWYQQK PGKAPKLLIY NAKTLAEGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ HHYGTPRTFG GGTKVEIKAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 34    Vector pure line: pIB1002 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRV TITCRASENI YSYLAWYQQK PGKAPKLLIY NAKTLAEGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ HHYGTPRTFG GGTKVEIKGG GGSGGGGSGG GGSQVQLVQS GAEVKKPGAS VKVSCKASGY TFTSYWMNWV RQAPGQGLEW MGRIDPYDSE THYAQKLQGR VTMTTDTSTS TAYMELRSLR SDDTAVYYCA RGGYDFDVGT LYWFFDVWGQ GTTVTVSSAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 35    Vector pure line: pIB1003 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVESGG GVVQPGRSLR LSCAASGFTF SSYDMHWVRQ APGKGLEWVA VIWYDGSNKY YADSVKGRFT ISRDNSKNTL YLQMNSLRAE DTAVYYCARG SGNWGFFDYW GQGTLVTVSS GGGGSGGGGS GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQGISRWLAW YQQKPEKAPK SLIYAASSLQ SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCQQYNTYP RTFGQGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 36    Vector pure line: pIB1004 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRV TITCRASQGI SRWLAWYQQK PEKAPKSLIY AASSLQSGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QYNTYPRTFG QGTKVEIKGG GGSGGGGSGG GGSQVQLVES GGGVVQPGRS LRLSCAASGF TFSSYDMHWV RQAPGKGLEW VAVIWYDGSN KYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCA RGSGNWGFFD YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 37    Vector pure line: pIB1005 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLQQWGA GLLKPSETLS LTCAVYGGSF SGYYWSWIRQ SPEGLEWIGE INHGGYVTYN PSLESRVTIS VDTSKNQFSL KLSSVTAADT AVYYCARDYG PGNYDWYFDL WGRGTLVTVS SGGGGSGGGG SGGGGSEIVL TQSPATLSLS PGERATLSCR ASQSVSSYLA WYQQKPGQAP RLLIYDASNR ATGIPARFSG SGSGTDFTLT ISSLEPEDFA VYYCQQRSNW PPALTFGGGT KVEIKRAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYN LFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESV TFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 38    Vector pure line: pIB1006 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPA TLSLSPGERA TLSCRASQSV SSYLAWYQQK PGQAPRLLIY DASNRATGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRSNWPPALT FGGGTKVEIK RGGGGSGGGG SGGGGSQVQL QQWGAGLLKP SETLSLTCAV YGGSFSGYYW SWIRQSPEGL EWIGEINHGG YVTYNPSLES RVTISVDTSK NQFSLKLSSV TAADTAVYYC ARDYGPGNYD WYFDLWGRGT LVTVSSAAAG SGGSGILVKQ SPMLVAYDNA VNLSCKYSYN LFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFN CDGKLGNESV TFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHV KGKHLCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 39    Vector pure line: pIB1007 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVTLRESGP ALVKPTQTLT LTCTFSGFSL STSGMGVGWI RQPPGKALEW LAHIWWDDDK YYNPSLKSRL TISKDTSKNQ VVLTMTNMDP VDTATYYCAR TRRYFPFAYW GQGTLVTVSS GGGGSGGGGS GGGGSEIVMT QSPATLSVSP GERATLSCKA SQNVGTNVAW YQQKPGQAPR LLIYSASYRY SGIPARFSGS GSGTEFTLTI SSLQSEDFAV YYCQQYNTDP LTFGGGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 40    Vector pure line: pIB1008 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEIVMTQSPA TLSVSPGERA TLSCKASQNV GTNVAWYQQK PGQAPRLLIY SASYRYSGIP ARFSGSGSGT EFTLTISSLQ SEDFAVYYCQ QYNTDPLTFG GGTKVEIKGG GGSGGGGSGG GGSQVTLRES GPALVKPTQT LTLTCTFSGF SLSTSGMGVG WIRQPPGKAL EWLAHIWWDD DKYYNPSLKS RLTISKDTSK NQVVLTMTNM DPVDTATYYC ARTRRYFPFA YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 41    Vector pure line: pIB1009 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGV EVKKPGASVK VSCKASGYTF TNYYMYWVRQ APGQGLEWMG GINPSNGGTN FNEKFKNRVT LTTDSSTTTA YMELKSLQFD DTAVYYCARR DYRFDMGFDY WGQGTTVTVS SGGGGSGGGG SGGGGSEIVL TQSPATLSLS PGERATLSCR ASKGVSTSGY SYLHWYQQKP GQAPRLLIYL ASYLESGVPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQH SRDLPLTFGG GTKVEIKRAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 42    Vector pure line: pIB1010 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPA TLSLSPGERA TLSCRASKGV STSGYSYLHW YQQKPGQAPR LLIYLASYLE SGVPARFSGS GSGTDFTLTI SSLEPEDFAV YYCQHSRDLP LTFGGGTKVE IKRGGGGSGG GGSGGGGSQV QLVQSGVEVK KPGASVKVSC KASGYTFTNY YMYWVRQAPG QGLEWMGGIN PSNGGTNFNE KFKNRVTLTT DSSTTTAYME LKSLQFDDTA VYYCARRDYR FDMGFDYWGQ GTTVTVSSAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 43    Vector pure line: pIB1011 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEVQLVESGG GLVQPGGSLR LSCAASGFTF SDSWIHWVRQ APGKGLEWVA WISPYGGSTY YADSVKGRFT ISADTSKNTA YLQMNSLRAE DTAVYYCARR HWPGGFDYWG QGTLVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QDVSTAVAWY QQKPGKAPKL LIYSASFLYS GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQYLYHPA TFGQGTKVEI KRAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 44    Vector pure line: pIB1012 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRV TITCRASQDV STAVAWYQQK PGKAPKLLIY SASFLYSGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QYLYHPATFG QGTKVEIKRG GGGSGGGGSG GGGSEVQLVE SGGGLVQPGG SLRLSCAASG FTFSDSWIHW VRQAPGKGLE WVAWISPYGG STYYADSVKG RFTISADTSK NTAYLQMNSL RAEDTAVYYC ARRHWPGGFD YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 45    Vector pure line: pIB1013 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGA EVKKPGASVK VSCKASGYTF TNYWIGWVKQ APGQGLEWIG YLYPGGLYTN YNEKFKGKAT MTADTSTNTA YMELSSLRSE DTAVYYCARY RDYDYAMDYW GQGTLVTVSS GGGGSGGGGS GGGGSDVVMT QTPLSLPVTL GQPASISCKS TKSLLNSDGF TYLGWCLQKP GQSPQLLIYL VSNRFSGVPD RFSGSGSGTD FTLKISRVEA EDVGVYYCFQ SNYLPLTFGQ GTKLEIKRAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 46    Vector pure line: pIB1014 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDVVMTQTPL SLPVTLGQPA SISCKSTKSL LNSDGFTYLG WCLQKPGQSP QLLIYLVSNR FSGVPDRFSG SGSGTDFTLK ISRVEAEDVG VYYCFQSNYL PLTFGQGTKL EIKRGGGGSG GGGSGGGGSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YWIGWVKQAP GQGLEWIGYL YPGGLYTNYN EKFKGKATMT ADTSTNTAYM ELSSLRSEDT AVYYCARYRD YDYAMDYWGQ GTLVTVSSAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 47    Vector pure line: pIB1015 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLQESGP GLVKPSQTLS LTCAVYGGSF SSGYWNWIRK HPGKGLEYIG YISYNGITYH NPSLKSRITI NRDTSKNQYS LQLNSVTPED TAVYYCARYK YDYDGGHAMD YWGQGTLVTV SSGGGGSGGG GSGGGGSDIQ MTQSPSSLSA SVGDRVTITC RASQDISNYL NWYQQKPGKA PKLLIYYTSK LHSGVPSRFS GSGSGTDYTL TISSLQPEDF ATYYCQQGSA LPWTFGQGTK VEIKAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 48    Vector pure line: pIB1016 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRV TITCRASQDI SNYLNWYQQK PGKAPKLLIY YTSKLHSGVP SRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGSALPWTFG QGTKVEIKGG GGSGGGGSGG GGSQVQLQES GPGLVKPSQT LSLTCAVYGG SFSSGYWNWI RKHPGKGLEY IGYISYNGIT YHNPSLKSRI TINRDTSKNQ YSLQLNSVTP EDTAVYYCAR YKYDYDGGHA MDYWGQGTLV TVSSAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 49    Vector pure line: pIB1017 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVESGG GVVQPGRSLR LSCAASGFTF SSYTMHWVRQ APGKGLEWVT FISYDGNNKY YADSVKGRFT ISRDNSKNTL YLQMNSLRAE DTAIYYCART GWLGPFDYWG QGTLVTVSSG GGGSGGGGSG GGGSEIVLTQ SPGTLSLSPG ERATLSCRAS QSVGSSYLAW YQQKPGQAPR LLIYGAFSRA TGIPDRFSGS GSGTDFTLTI SRLEPEDFAV YYCQQYGSSP WTFGQGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 50    Vector pure line: pIB1018 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPG TLSLSPGERA TLSCRASQSV GSSYLAWYQQ KPGQAPRLLI YGAFSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSPWTF GQGTKVEIKG GGGSGGGGSG GGGSQVQLVE SGGGVVQPGR SLRLSCAASG FTFSSYTMHW VRQAPGKGLE WVTFISYDGN NKYYADSVKG RFTISRDNSK NTLYLQMNSL RAEDTAIYYC ARTGWLGPFD YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 51    Vector pure line: pIB1019 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEVQLVESGG GVVRPGGSLR LSCVASGVTF DDYGMSWVRQ APGKGLEWVS GINWNGGDTD YSDSVKGRFT ISRDNAKNSL YLQMNSLRAE DTALYYCARD FYGSGSYYHV PFDYWGQGIL VTVSSGGGGS GGGGSGGGGS EIVLTQSPGT LSLSPGERAT LSCRASQSVS RSYLAWYQQK RGQAPRLLIY GASSRATGIP DRFSGDGSGT DFTLSISRLE PEDFAVYYCH QYDMSPFTFG PGTKVDIKAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 52    Vector pure line: pIB1020 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPG TLSLSPGERA TLSCRASQSV SRSYLAWYQQ KRGQAPRLLI YGASSRATGI PDRFSGDGSG TDFTLSISRL EPEDFAVYYC HQYDMSPFTF GPGTKVDIKG GGGSGGGGSG GGGSEVQLVE SGGGVVRPGG SLRLSCVASG VTFDDYGMSW VRQAPGKGLE WVSGINWNGG DTDYSDSVKG RFTISRDNAK NSLYLQMNSL RAEDTALYYC ARDFYGSGSY YHVPFDYWGQ GILVTVSSAA AGSGGSGILV KQSPMLVAYD NAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTG FNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 53    Vector pure line: pIB1021 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVESGG GVVQPGRSLR LSCAASGFSF SSTYVCWVRQ APGKGLEWIA CIYTGDGTNY SASWAKGRFT ISKDSSKNTV YLQMNSLRAE DTAVYFCARP DITYGFAINF WGPGTLVTVS SGGGGSGGGG SGGGGSDIQM TQSPSSLSAS VGDRVTIKCQ ASQSISSRLA WYQQKPGKPP KLLIYRASTL ASGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQCTGYG ISWPIGGGTK VEIKAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 54    Vector pure line: pIB1022 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRV TIKCQASQSI SSRLAWYQQK PGKPPKLLIY RASTLASGVP SRFSGSGSGT DFTLTISSLQ PEDVATYYCQ CTGYGISWPI GGGTKVEIKG GGGSGGGGSG GGGSQVQLVE SGGGVVQPGR SLRLSCAASG FSFSSTYVCW VRQAPGKGLE WIACIYTGDG TNYSASWAKG RFTISKDSSK NTVYLQMNSL RAEDTAVYFC ARPDITYGFA INFWGPGTLV TVSSAAAGSG GSGILVKQSP MLVAYDNAVN LSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCD GKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 55    Vector pure line: pIB1023 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGA EVKKPGASVK VSCKASGYTF TGYYMHWVRQ APGQGLEWMG WINPDSGGTN YAQKFQGRVT MTRDTSISTA YMELNRLRSD DTAVYYCARD QPLGYCTNGV CSYFDYWGQG TLVTVSSGGG GSGGGGSGGG GSDIQMTQSP SSVSASVGDR VTITCRASQG IYSWLAWYQQ KPGKAPNLLI YTASTLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQANIFPLTF GGGTKVEIKA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKY SYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGN ESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 56    Vector pure line: pIB1024 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SVSASVGDRV TITCRASQGI YSWLAWYQQK PGKAPNLLIY TASTLQSGVP SRFSGSGSGT DFTLTISSLQ PEDFATYYCQ QANIFPLTFG GGTKVEIKGG GGSGGGGSGG GGSQVQLVQS GAEVKKPGAS VKVSCKASGY TFTGYYMHWV RQAPGQGLEW MGWINPDSGG TNYAQKFQGR VTMTRDTSIS TAYMELNRLR SDDTAVYYCA RDQPLGYCTN GVCSYFDYWG QGTLVTVSSA AAGSGGSGIL VKQSPMLVAY DNAVNLSCKY SYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKT GFNCDGKLGN ESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 57    Vector pure line: pIB1025 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 58    Vector pure line: pIB1026 sequence: MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYQRSKKV AKKPTNKAPH PKQEPQELVETLPQ GDLPGGSNTA SEQ ID NO: 59    Vector pure line: pIB1027 sequence: MQSGTHWRVL GLCLLSVGVW GQDYKDDDDK DGNEEMGGIT QTPYKVSISG TTVILTCPQY PGSEILWQHN DKNIGGDEDD KNIGSDEDHL SLKEFSELEQ SGYYVCYPRG SKPEDANFYL YLRARVCENC MEMDVMSVAT IVIVDICITG GLLLLVYYWS RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 60    Vector pure line: pIB1028 sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDG SVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 61    Vector pure line: pIB1029 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQEQKLIS EEDL SEQ ID NO: 62    Vector pure line: pIB1030 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LSQPHTKPSV FVMKNGTNVA CLVKEFYPKD IRINLVSSKK ITEFDPAIVI SPSGKYNAVK LGKYEDSNSV TCSVQHDNKT VHSTDFEVKT DSTDHVKPKE TENTKQPSKS CHKPKAIVHT EKVNMMSLTV LGLRMLFAKT VAVNFLLTAK LFFLRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 63    Vector pure line: pIB1031 sequence: MALPVTALLL PLALLLHAAR PDYKDDDDKD KQLDADVSPK PTIFLPSIAE TKLQKAGTYL CLLEKFFPDV IKIHWQEKKS NTILGSQEGN TMKTNDTYMK FSWLTVPEKS LDKEHRCIVR HENNKNGVDQ EIIFPPIKTD VITMDPKDNC SKDANDTLLL QLTNTSAYYM YLLLLLKSVV YFAIITCCLL RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 64    Vector pure line: pIB1032 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LIQNPEPAVY QLKDPRSQDS TLCLFTDFDS QINVPKTMES GTFITDKCVL DMKAMDSKSN GAIAWSNQTS FTCQDIFKET NATYPSSDVP CDATLTEKSF ETDMNLNFQN LLVIVLRILL LKVAGFNLLM TLRLWSSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 65    Vector pure line: pIB1033 sequence: MALPVTALLL PLALLLHAAR PDYKDDDDKV LTPPKVSLFE PSKAEIANKQ KATLVCLARG FFPDHVELSW WVNGKEVHSG VCTDPQAYKE SNYSYCLSSR LRVSATFWHN PRNHFRCQVQ FHGLSEEDKW PEGSPKPVTQ NISAEAWGRA DCGITSASYQ QGVLSATILY EILLGKATLY AVLVSTLVVM AMVKRKNSRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q SEQ ID NO: 66    Vector pure line: pIB1046 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LSQPHTKPSV FVMKNGTNVA CLVKEFYPKD IRINLVSSKK ITEFDPAIVI SPSGKYNAVK LGKYEDSNSV TCSVQHDNKT VHSTDFEVKT DSTDHVKPKE TENTKQPSKS CHKPKAIVHT EKVNMMSLTV LGLRMLFAKT VAVNFLLTAK LFFLRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQRAK RGSGEGRGSL LTCGDVEENP GPMALPVTAL LLPLALLLHA ARPDYKDDDD KDKQLDADVS PKPTIFLPSI AETKLQKAGT YLCLLEKFFP DVIKIHWQEK KSNTILGSQE GNTMKTNDTY MKFSWLTVPE KSLDKEHRCI VRHENNKNGV DQEIIFPPIK TDVITMDPKD NCSKDANDTL LLQLTNTSAY YMYLLLLLKS VVYFAIITCC LLRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 67    Vector pure line: pIB1047 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LIQNPEPAVY QLKDPRSQDS TLCLFTDFDS QINVPKTMES GTFITDKCVL DMKAMDSKSN GAIAWSNQTS FTCQDIFKET NATYPSSDVP CDATLTEKSF ETDMNLNFQN LLVIVLRILL LKVAGFNLLM TLRLWSSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ RAKRGSGEGR GSLLTCGDVE ENPGPMALPV TALLLPLALL LHAARPDYKD DDDKVLTPPK VSLFEPSKAE IANKQKATLV CLARGFFPDH VELSWWVNGK EVHSGVCTDP QAYKESNYSY CLSSRLRVSA TFWHNPRNHF RCQVQFHGLS EEDKWPEGSP KPVTQNISAE AWGRADCGIT SASYQQGVLS ATILYEILLG KATLYAVLVS TLVVMAMVKR KNSRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 68    Vector pure line: pIB1048 sequence: MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQR AKRGSGEGRG SLLTCGDVEE NPGPMQSGTH WRVLGLCLLS VGVWGQDYKD DDDKDGNEEM GGITQTPYKV SISGTTVILT CPQYPGSEIL WQHNDKNIGG DEDDKNIGSD EDHLSLKEFS ELEQSGYYVC YPRGSKPEDA NFYLYLRARV CENCMEMDVM SVATIVIVDI CITGGLLLLV YYWSRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 69    Vector pure line: pIB1049 sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDG SVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ RAKRGSGMQS GTHWRVLGLC LLSVGVWGQD YKDDDDKDGN EEMGGITQTP YKVSISGTTV ILTCPQYPGS EILWQHNDKN IGGDEDDKNI GSDEDHLSLK EFSELEQSGY YVCYPRGSKP EDANFYLYLR ARVCENCMEM DVMSVATIVI VDICITGGLL LLVYYWSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 70    Vector pure line: pIB1050 sequence: MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFAGHET GRLSGAADTQ ALLRNDQVYQ PLRDRDDAQY SHLGGNWARN KRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQRAKRGS GEGRGSLLTC GDVEENPGPM QSGTHWRVLG LCLLSVGVWG QDYKDDDDKD GNEEMGGITQ TPYKVSISGT TVILTCPQYP GSEILWQHND KNIGGDEDDK NIGSDEDHLS LKEFSELEQS GYYVCYPRGS KPEDANFYLY LRARVCENCM EMDVMSVATI VIVDICITGG LLLLVYYWSK NRKAKAKPVT RGAGAGGRQR GQNKERPPPV PNPDYEPIRK GQRDLYSGLN QRRIRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 71    Vector pure line: pIB1051 sequence: MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFAGHET GRLSGAADTQ ALLRNDQVYQ PLRDRDDAQY SHLGGNWARN KRAKRGSGEG RGSLLTCGDV EENPGPMQSG THWRVLGLCL LSVGVWGQDY KDDDDKDGNE EMGGITQTPY KVSISGTTVI LTCPQYPGSE ILWQHNDKNI GGDEDDKNIG SDEDHLSLKE FSELEQSGYY VCYPRGSKPE DANFYLYLRA RVCENCMEMD VMSVATIVIV DICITGGLLL LVYYWSKNRK AKAKPVTRGA GAGGRQRGQN KERPPPVPNP DYEPIRKGQR DLYSGLNQRR I SEQ ID NO: 72    Vector pure line: pIB1052 sequence: MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARAKR GSGEGRGSLL TCGDVEENPG PMQSGTHWRV LGLCLLSVGV WGQDYKDDDD KDGNEEMGGI TQTPYKVSIS GTTVILTCPQ YPGSEILWQH NDKNIGGDED DKNIGSDEDH LSLKEFSELE QSGYYVCYPR GSKPEDANFY LYLRARVCEN CMEMDVMSVA TIVIVDICIT GGLLLLVYYW S SEQ ID NO: 73    Vector pure line: pIB1053 sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDG SVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIAGQD GVRQSRASDK QTLLPNDQLY QPLKDREDDQ YSHLQGNQLR RNRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQRAKRG SGEGRGSLLT CGDVEENPGP MQSGTHWRVL GLCLLSVGVW GQDYKDDDDK DGNEEMGGIT QTPYKVSISG TTVILTCPQY PGSEILWQHN DKNIGGDEDD KNIGSDEDHL SLKEFSELEQ SGYYVCYPRG SKPEDANFYL YLRARVCENC MEMDVMSVAT IVIVDICITG GLLLLVYYWS KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRRIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 74    Vector pure line: pIB1054 sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDG SVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIAGQD GVRQSRASDK QTLLPNDQLY QPLKDREDDQ YSHLQGNQLR RNRAKRGSGE GRGSLLTCGD VEENPGPMQS GTHWRVLGLC LLSVGVWGQD YKDDDDKDGN EEMGGITQTP YKVSISGTTV ILTCPQYPGS EILWQHNDKN IGGDEDDKNI GSDEDHLSLK EFSELEQSGY YVCYPRGSKP EDANFYLYLR ARVCENCMEM DVMSVATIVI VDICITGGLL LLVYYWSKNR KAKAKPVTRG AGAGGRQRGQ NKERPPPVPN PDYEPIRKGQ RDLYSGLNQR RI SEQ ID NO: 75    Vector pure line: pIB1055 sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDG SVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARAK RGSGEGRGSL LTCGDVEENP GPMQSGTHWR VLGLCLLSVG VWGQDYKDDD DKDGNEEMGG ITQTPYKVSI SGTTVILTCP QYPGSEILWQ HNDKNIGGDE DDKNIGSDED HLSLKEFSEL EQSGYYVCYP RGSKPEDANF YLYLRARVCE NCMEMDVMSV ATIVIVDICI TGGLLLLVYY WS SEQ ID NO: 76    Vector pure line: 77 pIB1056 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPRRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQEQK LISEEDL SEQ ID NO: 78    Vector pure line: pIB1057 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPREQKLIS EEDL SEQ ID NO: 79    Vector pure line: pIB1058 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LEQKLISEED L SEQ ID NO: 80    Vector pure line: pIB1059 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPRRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLD KRRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLST ATKDTYDALH MQALPPRRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ EQKLISEEDL SEQ ID NO: 81    Vector pure line: pIB1060 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPRRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLD KRRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLST ATKDTYDALH MQALPPREQK LISEEDL SEQ ID NO: 82    Vector pure line: pIB1061 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLD KRRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLST ATKDTYDALH MQALPPRRVK FSRSADAPAY QQGQNQLYNE LNLGRREEYD VLDKRRGRDP EMGGKPQRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR EQKLISEEDL SEQ ID NO: 83    Vector pure line: pIB1062 sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLD KRRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLST ATKDTYDALH MQALPPREQK LISEEDL SEQ ID NO: 84    Vector pure line: pIB1063 sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DNLLPSWAIT LISVNGIFVI CCL SEQ ID NO: 85    Vector pure line: pIB1064 sequence: MGCGCSSHPE DDWMENIDVC ENCHYPIVPL DGKGTLLIRN GSEVRDPLVT YEGSNPPASP LQDNLVIALH SYEPSHDGDL GFEKGEQLRI LEQSGEWWKA QSLTTGQEGF IPFNFVAKAN SLEPEPWFFK NLSRKDAERQ LLAPGNTHGS FLIRESESTA GSFSLSVRDF DQNQGEVVKH YKIRNLDNGG FYISPRITFP GLHELVRHYT NASDGLCTRL SRPCQTQKPQ KPWWEDEWEV PRETLKLVER LGAGQFGEVW MGYYNGHTKV AVKSLKQGSM SPDAFLAEAN LMKQLQHQRL VRLYAVVTQE PIYIITEYME NGSLVDFLKT PSGIKLTINK LLDMAAQIAE GMAFIEERNY IHRDLRAANI LVSDTLSCKI ADFGLARLIE DNEYTAREGA KFPIKWTAPE AINYGTFTIK SDVWSFGILL TEIVTHGRIP YPGMTNPEVI QNLERGYRMV RPDNCPEELY QLMRLCWKER PEDRPTFDYL RSVLEDFFTA TEGQYQPQPE QKLISEEDL SEQ ID NO: 86    Vector pure line: pIB1065 sequence: MGCGCSSHPE DDWMENIDVC ENCHYPIVPL DGKGTLLIRN GSEVRDPLVT YEGSNPPASP LQDNLVIALH SYEPSHDGDL GFEKGEQLRI LEQSGEWWKA QSLTTGQEGF IPFNFVAKAN SLEPEPWFFK NLSRKDAERQ LLAPGNTHGS FLIRESESTA GSFSLSVRDF DQNQGEVVKH YKIRNLDNGG FYISPRITFP GLHELVRHYT NASDGLCTRL SRPCQTQKPQ KPWWEDEWEV PRETLKLVER LGAGQFGEVW MGYYNGHTKV AVKSLKQGSM SPDAFLAEAN LMKQLQHQRL VRLYAVVTQE PIYIITEYME NGSLVDFLKT PSGIKLTINK LLDMAAQIAE GMAFIEERNY IHRDLRAANI LVSDTLSCKI ADFGLARLIE DNEYTAREGA KFPIKWTAPE AINYGTFTIK SDVWSFGILL TEIVTHGRIP YPGMTNPEVI QNLERGYRMV RPDNCPEELY QLMRLCWKER PEDRPTFDYL RSVLEDFFTA TEGQFQPQPE QKLISEEDL SEQ ID NO: 87    Vector pure line: pIB1066 sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIV PLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDG DLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWF FKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVV KHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQK PQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQG SMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFL KTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSC KIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGI LLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWK ERPEDRPTFD YLRSVLEDFF TATEGQYQPQ P SEQ ID NO: 88    Vector pure line: pIB1067 sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIV PLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDG DLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWF FKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVV KHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQK PQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQG SMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFL KTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSC KIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGI LLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWK ERPEDRPTFD YLRSVLEDFF TATEGQYQPQ PRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 89    Vector pure line: pIB1068 sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIV PLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDG DLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWF FKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVV KHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQK PQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQG SMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFL KTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSC KIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGI LLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWK ERPEDRPTFD YLRSVLEDFF TATEGQFQPQ P SEQ ID NO: 90    Vector pure line: pIB1069 sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIV PLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDG DLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWF FKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVV KHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQK PQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQG SMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFL KTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSC KIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGI LLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWK ERPEDRPTFD YLRSVLEDFF TATEGQFQPQ PRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 91    Vector pure line: pIB1070 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LMEEAILVPC VLGLLLLPIL AMLMALCVHC HRLPGSYDST SSDSLYPRGI QFKRPHTVAP WPPAYPPVTS YPPLSQPDLL PIPRSPQPLG GSHRTPSSRR DSDGANSVAS YENEGASGIR GAQAGWGVWG PSWTRLTPVS LPPEPACEDA DEDEDDYHNP GYLVVLPDST PATSTAAPSA PALSTPGIRD SAFSMESIDD YVNVPESGES AEASLDGSRE YVNVSQELHP GAAKTEPAAL SSQEAEEVEE EGAPDYENLQ ELN SEQ ID NO: 92    Vector pure line: pIB1071 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LMEEAILVPC VLGLLLLPIL AMLMALCVHC HRLPGSYDST SSDSLYPRGI QFKRPHTVAP WPPAYPPVTS YPPLSQPDLL PIPRSPQPLG GSHRTPSSRR DSDGANSVAS YENEGASGIR GAQAGWGVWG PSWTRLTPVS LPPEPACEDA DEDEDDYHNP GYLVVLPDST PATSTAAPSA PALSTPGIRD SAFSMESIDD YVNVPESGES AEASLDGSRE YVNVSQELHP GAAKTEPAAL SSQEAEEVEE EGAPDYENLQ ELNRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 93    Vector pure line: pIB1072 sequence: MNRGVPFRHL LLVLQLALLP AATQGEQKLI SEEDLKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWW QAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQ YAGSGNLTLA LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLML SLKLENKEAK VSKREKAVWV LNPEAGMWQC LLSDSGQVLL ESNIKVLPTW STPVQPMALI VLGGVAGLLL FIGLGIFFCV RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPI SEQ ID NO: 94    Vector pure line: pIB1073 sequence: MNRGVPFRHL LLVLQLALLP AATQGEQKLI SEEDLKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWW QAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQ YAGSGNLTLA LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLML SLKLENKEAK VSKREKAVWV LNPEAGMWQC LLSDSGQVLL ESNIKVLPTW STPVQPMALI VLGGVAGLLL FIGLGIFFCV RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPIRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q SEQ ID NO: 95    Vector pure line: pIB1074 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYVRAKRG SGEGRGSLLT CGDVEENPGP MRPRLWLLLA AQLTVLHGNS VDYKDDDDKL QQTPAYIKVQ TNKMVMLSCE AKISLSNMRI YWLRQRQAPS SDSHHEFLAL WDSAKGTIHG EEVEQEKIAV FRDASRFILN LTSVKPEDSG IYFCMIVGSP ELTFGKGTQL SVVDFLPTTA QPTKKSTLKK RVCRLPRPET QKGPLCSPIT LGLLVAGVLV LLVSLGVAIH LCCRRRRARL RFMKQFYK SEQ ID NO: 96    Vector pure line: pIB1075 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQRA KRGSGEGRGS LLTCGDVEEN PGPMRPRLWL LLAAQLTVLH GNSVDYKDDD DKLQQTPAYI KVQTNKMVML SCEAKISLSN MRIYWLRQRQ APSSDSHHEF LALWDSAKGT IHGEEVEQEK IAVFRDASRF ILNLTSVKPE DSGIYFCMIV GSPELTFGKG TQLSVVDFLP TTAQPTKKST LKKRVCRLPR PETQKGPLCS PITLGLLVAG VLVLLVSLGV AIHLCCRRRR ARLRFMKQFY KRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 97    Vector pure line: pIB1076 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LELTDTLQAE TDQLEDEKSA LQTEIANLLK EKEKLEFILA AHNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLVV ALGIGLFMRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP K PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TESAAPVQETLH GCQPVTQED SEQ ID NO: 98    Vector pure line: pIB1077 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LELTDTLQAE TDQLEDEKSA LQTEIANLLK EKEKLEFILA AHFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 99    Vector pure line: pIB1078 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LLEEKVKTLK AQNSELASTA NMLREQVAQL NCTYGCTGPG LEGCPTNGPK IPSIATGMVG ALLLLLVVAL GIGLFMRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 100    Vector pure line: pIB1079 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LLEEKVKTLK AQNSELASTA NMLREQVAQL FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 101    Vector pure line: pIB1080 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LETQHKVLEL TAENERLQKK VEQLSRELST NCTYGCTGPG LEGCPTNGPK IPSIATGMVG ALLLLLVVAL GIGLFMRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 102    Vector pure line: pIB1081 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LETQHKVLEL TAENERLQKK VEQLSRELST FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 103    Vector pure line: pIB1082 sequence: MRPSGTAGAA LLALLAALCP ASRAEQKLIS EEDLLEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLVV ALGIGLFMRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q SEQ ID NO: 104    Vector pure line: pIB1083 sequence: MRPSGTAGAA LLALLAALCP ASRAEQKLIS EEDLGTSGQK TKIISNRGEN SCKATGQVCH ALCSPEGCWG PEPRDCVSCR NVSRGRECVD KCNLLEGEPR EFVENSECIQ CHPECLPQAM NITCTGRGPD NCIQCAHYID GPHCVKTCPA GVMGENNTLV WKYADAGHVC HLCHPNCTYG CTGPGLEGCP TNGPKIPSIA TGMVGALLLL LVVALGIGLF MRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 105    Vector pure line: pIB1084 sequence: MRPSGTAGAA LLALLAALCP ASRAEQKLIS EEDLNCTYGC TGPGLEGCPT NGPKIPSIAT GMVGALLLLL VVALGIGLFM RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 106    Vector pure line: pIB1085 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 107    Vector pure line: pIB1086 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVEG ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 108    Vector pure line: pIB1087 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVD ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 109    Vector pure line: pIB1088 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVR ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 110    Vector pure line: pIB1089 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 111    Vector pure line: pIB1090 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVEG ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 112    Vector pure line: pIB1091 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVD ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 113    Vector pure line: pIB1092 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVR ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 114    Vector pure line: pIB1093 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVVG ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 115    Vector pure line: pIB1094 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVEG ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 116    Vector pure line: pIB1095 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVVD ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 117    Vector pure line: pIB1096 sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVVR ILLVVVLGVV FGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 118    Vector pure line: pIB1097 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQEQLVESGG RLVTPGTPLT LTCTASGFSL GSDFMSWVRQ APGKGLEWIG YIDPRSDIPY YASWAKGRFT ISKTSTTVDL KITSPTTEDT ATYFCARDLN AGYFNGIFYI WGPGTLVTVS SGGGGSGGGG SGGGGSELVM TQTPSSVSAA VGDTVTINCQ ASETVATLLA WYQQKPGQPP KLLIYGASNL ESGVPSRFRG SGSGTEFTLT ISGMKAEDAA TYYCQYGYIS TGSNTFGAGT NVEIKAAAGS GGSGILVKQS PMLVAYDNAV NLSCKYSYNL FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVT FYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 119    Vector pure line: pIB1098 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LELVMTQTPS SVSAAVGDTV TINCQASETV ATLLAWYQQK PGQPPKLLIY GASNLESGVP SRFRGSGSGT EFTLTISGMK AEDAATYYCQ YGYISTGSNT FGAGTNVEIK GGGGSGGGGS GGGGSQEQLV ESGGRLVTPG TPLTLTCTAS GFSLGSDFMS WVRQAPGKGL EWIGYIDPRS DIPYYASWAK GRFTISKTST TVDLKITSPT TEDTATYFCA RDLNAGYFNG IFYIWGPGTL VTVSSAAAGS GGSGILVKQS PMLVAYDNAV NLSCKYSYNL FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVT FYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 120    Vector pure line: pIB1099 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQEQLVESGG RLVTPGTPLT LTCTASGFSL GSDFMSWVRQ APGKGLEWIG YIDPRSDIPY YASWAKGRFT ISKTSTTVDL KITSPTTEDT ATYFCARDLN AGYFNGIFYI WGPGTLVTVS SGGGGSGGGG SGGGGSELDM TQTPSSTSEP VGGTVTINCQ ASQTISSYLS WYQQKPGHPP KLLIYDASDL ASGVPSRFSG SRSGTQFTLT ISGVQCDDAA TYYCLGVYDY RSDDGAAFGG GTELEILAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNES VTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 121    Vector pure line: pIB1100 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LELDMTQTPS STSEPVGGTV TINCQASQTI SSYLSWYQQK PGHPPKLLIY DASDLASGVP SRFSGSRSGT QFTLTISGVQ CDDAATYYCL GVYDYRSDDG AAFGGGTELE ILGGGGSGGG GSGGGGSQEQ LVESGGRLVT PGTPLTLTCT ASGFSLGSDF MSWVRQAPGK GLEWIGYIDP RSDIPYYASW AKGRFTISKT STTVDLKITS PTTEDTATYF CARDLNAGYF NGIFYIWGPG TLVTVSSAAA GSGGSGILVK QSPMLVAYDN AVNLSCKYSY NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGF NCDGKLGNES VTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 122    Vector pure line: pIB1101 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LQSLEESGGR LVTPGTPLTL TCTVSGFSLS TNDMNWVRQA PGKGLEWIGV IYSDDTPDYA TWAKGRFTIS RTSTTVDLKI TSPTTEDTAT YFCARGHYDS AVYAYALNIW GPGTLVTVSS GGGGSGGGGS GGGGSELVMT QTPSSVSAAV GGTVTITCQA SQSLSNLLAW YQQKPGQPPK LLIYGASNLE SGVPSRFRGS GSGTDFTLTI SGMKAEDAAT YYCQGGHYSG LTFGNGTNVE IKAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 123    Vector pure line: pIB1102 sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LELVMTQTPS SVSAAVGGTV TITCQASQSL SNLLAWYQQK PGQPPKLLIY GASNLESGVP SRFRGSGSGT DFTLTISGMK AEDAATYYCQ GGHYSGLTFG NGTNVEIKGG GGSGGGGSGG GGSQSLEESG GRLVTPGTPL TLTCTVSGFS LSTNDMNWVR QAPGKGLEWI GVIYSDDTPD YATWAKGRFT ISRTSTTVDL KITSPTTEDT ATYFCARGHY DSAVYAYALN IWGPGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLS CKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGK LGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 124    Vector pure line: pIB1103 sequence: MYGKIIFVLL LSEIVSISAE QKLISEEDLS STTGVAMHTS TSSSVTKSYI SSQTNDTHKR DTYAATPRAH EVSEISVRTV YPPEEETGER VQLAHHFSEP EITLIIFGVM AGVIGTILLI SYGRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QEDLHGCQPV TQ SEQ ID NO: 125    Vector pure line: pIB1104 sequence: MYGKIIFVLL LSEIVSISAE QKLISEEDLI TLIIFGVMAG VIGTILLISY GRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 126    Vector pure line: pIB1105 sequence: MDHLGASLWP QVGSLCLLLA GAAWEQKLIS EEDLAPPPNL PDPKFESKAA LLAARGPEEL LCFTERLEDL VCFWEEAASA GVGPGNYSFS YQLEDEPWKL CRLHQAPTAR GAVRFWCSLP TADTSSFVPL ELRVTAASGA PRYHRVIHIN EVVLLDAPVG LVARLADESG HVVLRWLPPP ETPMTSHIRY EVDVSAGNGA GSVQRVEILE GRTECVLSNL RGRTRYTFAV RARMAEPSFG GFWSAWSEPV SLLTPSDLDP LILTLSLILV VILVLLTVLA LLSRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 127    Vector pure line: pIB1106 sequence: MDHLGASLWP QVGSLCLLLA GAAWEQKLIS EEDLLILTLS LILVVILVLL TVLALLSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 128    Vector pure line: pIB1107 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALHL VLGLSAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 129    Vector pure line: pIB1108 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLSA VLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 130    Vector pure line: pIB1109 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALHL VLGLNAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 131    Vector pure line: pIB1110 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLNA VLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 132    Vector pure line: pIB1111 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALHL VLGLSAVLGL LLLRKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 133    Vector pure line: pIB1112 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLSA VLGLLLLRKR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 134    Vector pure line: pIB1113 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALLL VLGLSAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 135    Vector pure line: pIB1114 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALLLVLGLSA VLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 136    Vector pure line: pIB1115 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALHL VLGLNAVLGL LLLRKRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 137    Vector pure line: pIB1116 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLNA VLGLLLLRKR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 138    Vector pure line: pIB1117 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTALYL VLGLNAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 139    Vector pure line: pIB1118 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALYLVLGLNA VLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 140    Vector pure line: pIB1119 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKC FSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTR YVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIK AMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIAT ETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGS CLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQ CFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKT NPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVR LPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEP PLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETA WISLVTAWCL VLGLSAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 141    Vector pure line: pIB1120 sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT AWCLVLGLSA VLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 132    Vector pure line: pIB1179 sequence: MALPVTALLL PLALLLHAAR PEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKAAAFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 143    Vector pure line: pIB1180 sequence: MALPVTALLL PLALLLHAAR PAEPKSPDKT HTCPPCPAPP VAGPSVFLFP PKPKDTLMIA RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKKDPKFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 144    Vector pure line: pIB1181 sequence: MALPVTALLL PLALLLHAAR PERKCCVECP PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIS VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 145    Vector pure line: pIB1182 sequence: MALPVTALLL PLALLLHAAR PELKTPLGDT THTCPRCPEP KSCDTPPPCP RCPEPKSCDT PPPCPRCPEP KSCDTPPPCP RCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVQFKW YVDGVEVHNA KTKPREEQYN STFRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KTKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESSGQP ENNYNTTPPM LDSDGSFFLY SKLTVDKSRW QQGNIFSCSV MHEALHNRFT QKSLSLSPGK FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 146    Vector pure line: pIB1183 sequence: MALPVTALLL PLALLLHAAR PESKYGPPCP SCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 147    Vector pure line: pIB1184 sequence: MALPVTALLL PLALLLHAAR PESKYGPPCP PCPAPEFEGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFQ STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 148    Vector pure line: pIB1185 sequence: MALPVTALLL PLALLLHAAR PEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKAAAIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 149    Vector pure line: pIB1186 sequence: MALPVTALLL PLALLLHAAR PAEPKSPDKT HTCPPCPAPP VAGPSVFLFP PKPKDTLMIA RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKKDPKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 150    Vector pure line: pIB1187 sequence: MALPVTALLL PLALLLHAAR PERKCCVECP PCPAPPVAGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TFRVVSVLTV VHQDWLNGKE YKCKVSNKGL PAPIEKTISK TKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIS VEWESNGQPE NNYKTTPPML DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 151    Vector pure line: pIB1188 sequence: MALPVTALLL PLALLLHAAR PELKTPLGDT THTCPRCPEP KSCDTPPPCP RCPEPKSCDT PPPCPRCPEP KSCDTPPPCP RCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVQFKW YVDGVEVHNA KTKPREEQYN STFRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KTKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESSGQP ENNYNTTPPM LDSDGSFFLY SKLTVDKSRW QQGNIFSCSV MHEALHNRFT QKSLSLSPGK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 152    Vector pure line: pIB1189 sequence: MALPVTALLL PLALLLHAAR PESKYGPPCP SCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 153    Vector pure line: pIB1190 sequence: MALPVTALLL PLALLLHAAR PESKYGPPCP PCPAPEFEGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFQ STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 154    logo CD40 sequence KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 155    logo CD40_tandem sequence KKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 156    logo CD40_P227A sequence KKVAKKPTNK AAHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The invention will be further illustrated in the following examples, which are given for illustrative purposes only and are not intended to limit the invention in any way. example

Co-culture assay setup. T cells from 2 healthy donors were modified to express the tested or maintained non-transduced (NTD) constructs at MOI 10. The day before the co-culture setup, effector T cells were thawed and resuspended in T cell medium (TCM) at 1 x ¹⁰ cells/ml in the absence of IL2 and incubated at 37°C with 5% _CO overnight. On the day of co-culture, T cells (effectors) and Ba/F3-OKT3 targets were collected and counted using ViCELL BLU according to the manufacturer's instructions. T cells were then co-cultured overnight with the Ba/F3 OKT3 target at 10:1, 1:1 and 1:10 E:T (effector:target) ratios. For the inducible costimulatory protein constructs tested (ie, pIB1097 to pIB1102), an additional set of wells were set up in which, in addition to the Ba/F3 OKT3 target, pembrolizumab was also added at 10 μg/mL. Each condition was performed in duplicate. Unstimulated T cells served as negative controls. Two pools for each E:T ratio and a control plate of T cells only were set up. Brefeldin A was added at a 1 : 1000 dilution to a set of culture plates to assess intercellular production by intracellular intercellular staining (ICS) using flow cytometry after overnight co-cultivation. The second group was incubated for 5 days, after which T cell counts and expression of activation markers (ie, 41BB and CD69) were assessed by flow cytometry.

The present invention includes modifying components of the TCR complex and related signaling adaptors (such as in the TCR-incorporated antigen-indeterminate receptor "TIAAR"), identifying transmembrane domains (TMDs) and implementing receptors ("constitutive" "), and uses antibodies to induce receptor activation ("inducible").

scFv targeting costimulatory or inhibitory receptors and ligands. scFVs are derived from antibodies targeting co-stimulatory or inhibitory molecules expressed on immune cells. signal peptide Label ECD_TMD ICD Costim GOI description CD8a Myc hZ270_HL NA CD28-CD40 Anti-NKG2A blockade CD8a Myc hZ270_LH NA CD28-CD40 Anti-NKG2A blockade CD8a Myc Valimumab_HL NA CD28-CD40 anti-CD27 agonist CD8a Myc Valimumab_LH NA CD28-CD40 anti-CD27 agonist CD8a Myc Urilumab_HL NA CD28-CD40 anti-CD137 agonist CD8a Myc Urilumab_LH NA CD28-CD40 anti-CD137 agonist CD8a Myc TRX518_HL NA CD28-CD40 Anti-GITR Agonists CD8a Myc TRX518_LH NA CD28-CD40 Anti-GITR Agonists CD8a Myc Pembrolizumab_HL NA CD28-CD40 Anti-PD1 blockade CD8a Myc Pembrolizumab_LH NA CD28-CD40 Anti-PD1 blockade CD8a Myc Atezolizumab_HL NA CD28-CD40 Anti-PDL1 blockade CD8a Myc Atezolizumab_LH NA CD28-CD40 Anti-PDL1 blockade CD8a Myc RONK203_HL NA CD28-CD40 Anti-FasL blocking CD8a Myc RONK203_LH NA CD28-CD40 Anti-FasL blocking CD8a Myc Tacridezole_HL NA CD28-CD40 Anti-OX40 Agonist CD8a Myc Tacridezole_LH NA CD28-CD40 Anti-OX40 Agonist CD8a Myc Ipilimumab_HL NA CD28-CD40 Anti-CTLA4 blockade CD8a Myc Ipilimumab_LH NA CD28-CD40 Anti-CTLA4 blockade CD8a Myc KY1044_HL NA CD28-CD40 Anti-ICOS agonists CD8a Myc KY1044_LH NA CD28-CD40 Anti-ICOS agonists CD8a Myc APX005_HL NA CD28-CD40 anti-CD40 agonist CD8a Myc APX005_LH NA CD28-CD40 anti-CD40 agonist CD8a Myc Cerumumab_HL NA CD28-CD40 anti-CD40 agonist CD8a Myc Cerumumab_LH NA CD28-CD40 anti-CD40 agonist

TIAAR (incorporated into TCR) list of constructs code concept signal peptide Label ECD_TMD ICD Costim GOI description pIB1026 TIAAR CD3D Myc CD3D N/A CD28-CD40 CD3D_CD3D_CD28CD40 pIB1027 TIAAR CD3E FLAG CD3E N/A CD28-CD40 CD3E_CD3E_CD28CD40 pIB1028 TIAAR CD3G Myc CD3G N/A CD28-CD40 CD3G_CD3G_CD28CD40 pIB1029 TIAAR CD3Z Myc IC CD3Z N/A CD28-CD40 CD3Z_CD3Z_CD28CD40_Myc pIB1030 TIAAR CD8A Myc hTRDC N/A CD28-CD40 CD8A_hTRDC_CD28CD40 pIB1031 TIAAR CD8A FLAG hTRGC1 N/A CD28-CD40 CD8A_hTRGC1_CD28CD40 pIB1032 TIAAR CD8A Myc mTRAC N/A CD28-CD40 CD8A_mTRAC_CD28CD40 pIB1033 TIAAR CD8A FLAG mTRBC1 N/A CD28-CD40 CD8A_mTRBC1_CD28CD40 pIB1046 TIAAR CD8A x2 Myc and FLAG hTRDC_hTRGC1 N/A CD28-CD40 CD8A_hTRDC_CD28CD40-T2A-CD8a_hTRGC1_CD28CD40 pIB1047 TIAAR CD8A x2 Myc and FLAG mTRAC_mTRBC1 N/A CD28-CD40 CD8A_mTRAC_CD28CD40-T2A-CD8A_mTRBC1_CD28CD40 pIB1048 TIAAR CD3D and CD3E Myc and FLAG CD3D_CD3E N/A CD28-CD40 CD3D_CD3D_CD28CD40-T2A-CD3E_CD3E_CD28CD40 pIB1049 TIAAR CD3G and CD3E Myc and FLAG CD3G_CD3E N/A CD28-CD40 CD3G_CD3G_CD28CD40-T2A-CD3E_CD3E_CD28CD40 pIB1050 TIAAR CD3D and CD3E Myc and FLAG CD3D_CD3E CD3D_CD3E CD28-CD40 CD3D_CD3D_CD3D (ICD)_CD28CD40-T2A-CD3E_CD3E_CD3E (ICD)_CD28CD40 pIB1051 TIAAR CD3D and CD3E Myc and FLAG CD3D_CD3E CD3D_CD3E N/A CD3D_CD3D_CD3D ICD -T2A-CD3E_CD3E_CD3E ICD pIB1052 TIAAR CD3D and CD3E Myc and FLAG CD3D_CD3E N/A N/A CD3D_CD3D (control)-T2A-CD3E_CD3E (control) pIB1053 TIAAR CD3G and CD3E Myc and FLAG CD3G_CD3E CD3G_CD3E CD28-CD40 CD3G_CD3G_CD3G (ICD)_CD28CD40-T2A-CD3E_CD3E_CD3E (ICD)_CD28CD40 pIB1054 TIAAR CD3G and CD3E Myc and FLAG CD3G_CD3E CD3G_CD3E N/A CD3G_CD3G_CD3G ICD -T2A-CD3E_CD3E_CD3E ICD pIB1055 TIAAR CD3G and CD3E Myc and FLAG CD3G_CD3E N/A N/A CD3G_CD3G (control)-T2A-CD3E_CD3E (control) pIB1056 TIAAR CD3Z Myc IC CD3Z CD3Z CD28-CD40 CD3z _CD3z_CD3z ICD _CD28CD40_Myc pIB1057 TIAAR CD3Z Myc IC CD3Z CD3Z N/A CD3z _CD3z_CD3z ICD_Myc pIB1058 TIAAR CD3Z Myc IC CD3Z N/A N/A CD3z_CD3z (control) pIB1059 TIAAR CD3Z Myc IC CD3Z CD3Z (x2) CD28-CD40 CD3Z_CD3Z_CD3Z ICD (repeat CD3z intracellular domain-6 ITAMs) _CD28CD40_Myc pIB1060 TIAAR CD3Z Myc IC CD3Z CD3Z (x2) N/A CD3Z_CD3Z_CD3Z ICD (repeat CD3z intracellular domain-6 ITAMs)_Myc pIB1061 TIAAR CD3Z Myc IC CD3Z CD3Z (x2) CD28-CD40 (swap) CD3Z_CD3Z_CD28CD40_CD3Z (6 ITAMs)_Myc pIB1062 TIAAR CD3Z Myc IC CD3Z CD3Z CD28-CD40 (swap) CD3Z_CD3Z_CD28CD40_CD3Z ICD_Myc pIB1063 TIAAR CD80 Myc CD80 CD80 N/A CD80 (control) pIB1064 TIAAR No signal peptide Myc IC N/A Lck N/A Lck (control) pIB1065 TIAAR No signal peptide Myc IC N/A Lck (Y505F) N/A Lck (Y505F) (control) pIB1066 TIAAR CD80 Myc CD80 Lck N/A CD80_Lck pIB1067 TIAAR CD80 Myc CD80 Lck CD28-CD40 CD80_Lck_CD28CD40 pIB1068 TIAAR CD80 Myc CD80 CD80_Lck (Y505F) N/A CD80_Lck (Y505F) pIB1069 TIAAR CD80 Myc CD80 CD80_Lck (Y505F) CD28-CD40 CD80_Lck (Y505F)_CD28CD40 pIB1070 TIAAR CD8A Myc LAT LAT N/A LAT (control) pIB1071 TIAAR CD8A Myc LAT LAT CD28-CD40 LAT_C28CD40 pIB1072 TIAAR CD4 Myc CD4 CD4 CD28-CD40 CD4 control pIB1073 TIAAR CD4 Myc CD4 CD4 CD28-CD40 CD4_CD28_CD40 pIB1074 TIAAR CD8A and CD8B Myc and FLAG CD8A and CD8B CD8A and CD8B N/A CD8 control pIB1075 TIAAR CD8A and CD8B Myc and FLAG CD8A and CD8B CD8A and CD8B CD28-CD40 CD8_CD28_CD40

Cytokinin production (Bcl-xL, IL2, IFNγ) from genetically modified and untransduced T cells (NTDs) following stimulation with Ba/F3 OKT3 targets or remaining unstimulated (ie, T cells only) overnight and TNFα) (Figure 2). The production of Bcl-xL, IL2, IFNγ and TNFα from genetically modified T cells was increased compared to NTD cells in Donor 1. Compared to NTD cells in Donor 2, there was increased IFNγ production and comparable or lower production of Bcl-xL, IL2, IFNγ and TNFα in the genetically modified T cells.

Proliferation of genetically modified and untransduced T cells (NTD) (T cell counts from CD45+ (TIARR) after co-culture with Ba/F3 OKT3 targets or left unstimulated (ie T cells only) for 5 days ) and activation marker expression (41BB from CD45+ and CD69 from CD45+) (Figure 3). The genetically modified T cell counts were reduced compared to NTD cells in Donor 1. There was increased or similar expression of 41BB and CD69 in genetically modified T cells compared to NTD cells in Donor 1. Compared to NTD cells in Donor 1, there was increased or similar expression of 41BB and CD69 in genetically modified T cells. Compared to NTD cells in Donor 2, there were increased T cell counts for the 2 tested modifications, and similar or decreased T cell counts for the remaining genetically modified T cells. There was increased or comparable expression of 41BB and CD169 in genetically modified T cells compared to NTD cells in Donor 2.

List of Component Constructs code concept signal peptide Label ECD_TMD ICD Costim GOI description pIB1076 C-SAAR CD8A Myc LZ (cFos)_EGFR N/A CD28-CD40 LZ (cFos)-EGFRTM/JMD-CD28-CD40 pIB1077 C-SAAR CD8A Myc LZ (cFos)_CD28 N/A CD28-CD40 LZ (cFos)-CD28TM-CD28-CD40 pIB1078 C-SAAR CD8A Myc LZ (cJun)_EGFR N/A CD28-CD40 LZ (cJun)-EGFRTM/JMD-CD28-CD40 pIB1079 C-SAAR CD8A Myc LZ (cJun)_CD28 N/A CD28-CD40 LZ (cJun)-CD28TM-CD28-CD40 pIB1080 C-SAAR CD8A Myc LZ (c/EBP)_EGFR N/A CD28-CD40 LZ (c/EBP)-EGFRTM/JMD-CD28-CD40 pIB1081 C-SAAR CD8A Myc LZ (c/EBP)_CD28 N/A CD28-CD40 LZ(c/EBP)-CD28TM-CD28-CD40 pIB1103 C-SAAR GpA Myc GpA ECD_TMD N/A CD28-CD40 GpA ECD-TMD-CD28-CD40 pIB1104 C-SAAR GpA Myc GpA TMD N/A CD28-CD40 GpA TMD-CD28-CD40 pIB1105 C-SAAR EPOR Myc EPOR ECD_TMD N/A CD28-CD40 EpoR ECD-TMD-CD28-CD40 pIB1106 C-SAAR EPOR Myc EPORTM N/A CD28-CD40 EpoR TMD-CD28-CD40 pIB1107 C-SAAR TPOR Myc TPOR ECD_TMD N/A CD28-CD40 TPO ECD- TPO (WT) TMD-CD28-CD40 pIB1108 C-SAAR TPOR Myc TPOR TMD N/A CD28-CD40 TPO (WT) TMD-CD28-CD40 pIB1109 C-SAAR TPOR Myc TPOR ECD_TMD (S505N) N/A CD28-CD40 TPO ECD- TPO (S505N) TMD-CD28-CD40 pIB1110 C-SAAR TPOR Myc TPOR TMD (S505N) N/A CD28-CD40 TPO (S505N) TMD-CD28-CD40 pIB1111 C-SAAR TPOR Myc TPOR ECD_TMD (W515K) N/A CD28-CD40 TPO ECD- TPO (W515K) TMD-CD28-CD40 pIB1112 C-SAAR TPOR Myc TPOR TMD (W515K) N/A CD28-CD40 TPO (W515K) TMD-CD28-CD40 pIB1113 C-SAAR TPOR Myc TPOR ECD_TMD (H499L) N/A CD28-CD40 TPO ECD- TPO (H499L) TMD-CD28-CD40 pIB1114 C-SAAR TPOR Myc TPOR TMD (H499L) N/A CD28-CD40 TPO (H499L) TMD-CD28-CD40 pIB1115 C-SAAR TPOR Myc TPOR ECD_TMD (S505N-W515K) N/A CD28-CD40 TPO ECD- TPO (S505N-W515K) TMD-CD28-CD40 pIB1116 C-SAAR TPOR Myc TPOR TMD (S505N-W515K) N/A CD28-CD40 TPO (S505N-W515K) TMD-CD28-CD40 pIB1117 C-SAAR TPOR Myc TPOR ECD_TMD (H499Y-S505N) N/A CD28-CD40 TPO ECD- TPO (H499Y-S505N) TMD-CD28-CD40 pIB1118 C-SAAR TPOR Myc TPOR TMD (H499Y-S505N) N/A CD28-CD40 TPO (H499Y-S505N) TMD-CD28-CD40 pIB1119 C-SAAR TPOR Myc TPOR ECD_TMD (L498W-H499C) N/A CD28-CD40 TPO ECD-TPO (L498W-H499C) TMD-CD28-CD40 pIB1120 C-SAAR TPOR Myc TPOR TMD (L498W-H499C) N/A CD28-CD40 TPO (L498W-H499C) TMD-CD28-CD40 pIB1025 C-SAAR CD8a Myc CD28 N/A CD28-CD40 CD28TM_CD28_CD40 pIB1179 C-SAAR CD8a N/A IgG1+CD28TM N/A CD28-CD40 IgG1(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1180 C-SAAR CD8a N/A IgG1mut+CD28TM N/A CD28-CD40 IgG1(CH2CH3, mutant)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1181 C-SAAR CD8a N/A IgG2+CD28TM N/A CD28-CD40 IgG2(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1182 C-SAAR CD8a N/A IgG3+CD28TM N/A CD28-CD40 IgG3(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1183 C-SAAR CD8a N/A IgG4+CD28TM N/A CD28-CD40 IgG4(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1184 C-SAAR CD8a N/A IgG4mut+CD28TM N/A CD28-CD40 IgG4(CH2CH3, mutant)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1185 C-SAAR CD8a N/A IgG1 + CD28 STEM/TM N/A CD28-CD40 IgG1(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1186 C-SAAR CD8a N/A IgG1mut + CD28 STEM/TM N/A CD28-CD40 IgG1(CH2CH3, mutant)-CD28(STEM+TM)-CD28(CoStim)-CD40(CoStim) pIB1187 C-SAAR CD8a N/A IgG2 + CD28 STEM/TM N/A CD28-CD40 IgG2(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1188 C-SAAR CD8a N/A IgG3 + CD28 STEM/TM N/A CD28-CD40 IgG3(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1189 C-SAAR CD8a N/A IgG4 + CD28 STEM/TM N/A CD28-CD40 IgG4(CH2CH3)-CD28(Stem+TM)-CD28(CoStim)-CD40(CoStim) pIB1190 C-SAAR CD8a N/A IgG4mut + CD28 STEM/TM N/A CD28-CD40 IgG4(CH2CH3, mutant)-CD28(STEM+TM)-CD28(CoStim)-CD40(CoStim)

Cytokinin production (Bcl-xL, IL2, IFNγ) from genetically modified and untransduced T cells (NTDs) following stimulation with Ba/F3 OKT3 targets or remaining unstimulated (ie, T cells only) overnight and TNFα) (Figure 4). Compared to NTD cells in Donor 1, there was increased or comparable Bcl-xL, IL2 and TNFα production and comparable or lower IFNγ content in the genetically modified T cells. Compared to NTD cells in Donor 2, there was increased IFNγ and IL2 production and comparable or lower Bcl-xL and TNFα production in the genetically modified T cells.

Proliferation of genetically modified and untransduced T cells (NTD) (T cell counts from CD45+ (LZ) after co-culture with Ba/F3 OKT3 targets or left unstimulated (ie T cells only) for 5 days ) and activation marker expression (41BB from CD45+ and CD69 from CD45+) (Figure 5). The genetically modified T cell counts were reduced compared to NTD cells in Donor 1. There was increased expression of 41BB and CD69 in genetically modified T cells compared to NTD cells in Donor 1. T cell counts were increased compared to NTD cells in Donor 2, 41BB expression was increased in GM T cells compared to NTD cells, and GM T cells were increased compared to NTD cells in Donor 2. CD169 expression was similar or decreased.

List of Inductive Constructs code concept signal peptide Label ECD_TMD ICD Costim GOI description pIB1082 Inducible EGFR Myc EGFR N/A CD28-CD40 WT EGFR ECD-EGFRTM/JMD-CD28-CD40 pIB1083 Inducible EGFR Myc EGFR (Domain IV) N/A CD28-CD40 Domain IV - EGFRTM/JMD-CD28-CD40 pIB1084 Inducible EGFR Myc EGFR (623-668) N/A CD28-CD40 EGFRTM/JMD-CD28-CD40 (control) pIB1085 Inducible Her2 Myc Her2 N/A CD28-CD40 Her 2 (Domains 1 to IV)-TMD/JMD-CD28-CD40 pIB1086 Inducible Her2 Myc Her2 (V659E) N/A CD28-CD40 Her 2 (Domains 1 to IV)-TMD (V659E)/JMD-CD28-CD40 pIB1087 Inducible Her2 Myc Her2 (V660D) N/A CD28-CD40 Her 2 (Domains 1 to IV)-TMD (G660D)/JMD-CD28-CD40 pIB1088 Inducible Her2 Myc Her2 (V660R) N/A CD28-CD40 Her 2 (Domains 1 to IV)-TMD (G660R)/JMD-CD28-CD40 pIB1089 Inducible Her2 Myc Her2 Domain IV_TMD N/A CD28-CD40 Her 2 (Domain IV)-TMD/JMD-CD28-CD40 pIB1090 Inducible Her2 Myc Her2 Domain IV_TMD (V659E) N/A CD28-CD40 Her 2 (Domain IV)-TMD (V659E)/JMD-CD28-CD40 pIB1091 Inducible Her2 Myc Her2 Domain IV_TMD (G660D) N/A CD28-CD40 Her 2 (Domain IV)-TMD (G660D)/JMD-CD28-CD40 pIB1092 Inducible Her2 Myc Her2 Domain IV_TMD (G660R) N/A CD28-CD40 Her 2 (Domain IV)-TMD (G660R)/JMD-CD28-CD40 pIB1093 Inducible Her2 Myc Her2 TMD N/A CD28-CD40 Her 2 TMD/JMD-CD28-CD40 pIB1094 Inducible Her2 Myc Her2 TMD (V659E) N/A CD28-CD40 Her 2 TMD (V659E)/JMD-CD28-CD40 pIB1095 Inducible Her2 Myc Her2 TMD (G660D) N/A CD28-CD40 Her 2 TMD (G660D)/JMD-CD28-CD40 pIB1096 Inducible Her2 Myc Her2 TMD (G660R) N/A CD28-CD40 Her 2 TMD (G660R)/JMD-CD28-CD40 pIB1097 Inducible CD8A Myc A30514 VH_VL N/A CD28-CD40 Anti-ID1 VH-VL (A30514-Pembrolizumab)- CD28TMD CD28-CD40 pIB1098 Inducible CD8A Myc A30514 VL_VH N/A CD28-CD40 Anti-ID1 VL-VH (A30514-Pembrolizumab)- CD28TMD CD28-CD40 pIB1099 Inducible CD8A Myc A30523 VH_VL N/A CD28-CD40 Anti-ID2 Vh-VL (A30523-Pembrolizumab)- CD28TMD CD28-CD40 pIB1100 Inducible CD8A Myc A30523 VL_VH N/A CD28-CD40 Anti-ID2 VL-Vh (A30523-Pembrolizumab)- CD28TMD CD28-CD40 pIB1101 Inducible CD8A Myc A30633 VH_VL N/A CD28-CD40 Anti-ID3 Vh-VL (A30633-Pembrolizumab)- CD28TMD CD28-CD40 pIB1102 Inducible CD8A Myc A30633 VL_VH N/A CD28-CD40 Anti-ID3 VL-VH (A30633-Pembrolizumab)- CD28TMD CD28-CD40

Genetically modified and untransduced T after stimulation with Ba/F3 OKT3 target or Ba/F3 OKT3 target with 10 μg/mL pembrolizumab or left unstimulated (ie T cells only) overnight Cytokinin production (Bcl-xL, IL2, IFNg and TNFa) by cells (NTD) (Figure 6). Compared with conditions using Ba/F3 OKT3 OKT3 stimulation alone and NTD cells in Donor 1 and Donor 2, in the presence of Ba/F3 OKT3 and pembrolizumab, Bcl-xL, The production of IL2, IFNγ and TNFα was increased or comparable.

Genetically modified and untransduced after 5 days of co-culture with Ba/F3 OKT3 target or Ba/F3 OKT3 target with 10 μg/mL pembrolizumab or left unstimulated (ie T cells only) for 5 days Proliferation (count of T cells from CD45+ (inducible)) and expression of activation markers (41BB from CD45+ and CD69 from CD45+) in T cells (NTDs) (Figure 7). Compared to NTD cells in Donor 1, T cell counts in genetically modified T cells were increased, comparable and decreased. Compared to NTD cells in Donor 1, there was increased or similar expression of 41BB and CD169 in genetically modified T cells. The T cell counts in the genetically modified T cells were increased or comparable compared to the NTD cells in Donor 2. Compared to NTD cells in Donor 2, 41BB and CD169 expression was increased, similar and decreased in genetically modified cells. When genetically modified T cells were stimulated with Ba/F3 OKT3 in the presence of pembrolizumab compared to conditions stimulated with Ba/F3 OKT3 alone and NTD cells, increased T cell counts were observed in both donors and Activation marker performance. * * *

Having thus described in detail preferred embodiments of the invention, it is to be understood that the invention, as defined in the preceding paragraphs, is not limited to the specific details set forth in the foregoing description, for obvious variations thereof may be made without departing from the invention the spirit or scope of the situation.

Given by way of example, and not intended to limit the invention to the particular embodiments described, the following embodiments are best understood in conjunction with the accompanying drawings.

Figure 1 - Schematic model of a universal costimulatory protein. (A) TCR-incorporated antigen-indeterminate receptors (TIAARs) comprise modified components of the TCR complex and associated signaling adaptors. (B) Constitutive costimulatory receptors comprising a transmembrane domain (TMD) and features that allow for inducible or constitutive activation. (C) Inducible costimulatory receptors capable of induction and activation by extracellular ligand binding.

Figure 2 - Interferon production by cells transduced by TCR-incorporated antigen-defined receptor (TIAAR). Gene-modified and non-transduced T cells (NTDs) from both donors were assayed for cytokine production after overnight stimulation with the Ba/F3 OKT3 target or left unstimulated (ie, T cells only) (i.e., T cells only) ( Bcl-xL, IL2, IFNg and TNFa).

Figure 3 - Proliferation and activation marker expression of cells transduced by TIAAR. Genetically modified and untransduced cells from Donor 1 (3A) and Donor 2 (3B) were assayed after 5 days of co-culture with Ba/F3 OKT3 targets or left unstimulated (ie, T cells only) for 5 days Proliferation of T cells (NTD) (T cell count) and expression of activation markers (41BB and CD69).

Figure 4 - Interferon production in leucine zipper-based universal CoStAR (LZ) transduced cells. Genetically modified and untransduced T cells (NTDs) from both donors were assayed for cytokine production (Bcl -xL, IL2, IFNg and TNFa).

Figure 5 - Expression of proliferation and activation markers in cells transduced with LZ-CoStAR. Genetically modified and untransduced cells from Donor 1 (5A) and Donor 2 (5B) were assayed after 5 days of co-culture with Ba/F3 OKT3 targets or left unstimulated (ie T cells only) for 5 days Proliferation of T cells (NTD) (T cell count) and expression of activation markers (41BB and CD69).

Figure 6 - Cytokinin production in inducible universal CoStAR-transduced cells. Genetically modified and untransduced T cells (NTDs) from both donors were assayed for cytokine production (Bcl -xL, IL2, IFNg and TNFa). Generic CoStAR can be induced by pembrolizumab.

Figure 7 - Expression of proliferation and activation markers in cells transduced by inducible universal CoStAR. Genetically modified and untransduced cells from Donor 1 (7A) and Donor 2 (7B) were assayed after co-culturing with Ba/F3 OKT3 targets or remaining unstimulated (ie T cells only) for 5 days Proliferation of T cells (NTD) (T cell count) and expression of activation markers (41BB and CD69). Generic CoStAR can be induced by pembrolizumab.

Claims (45)

An engineered protein comprising a cluster domain and a messaging domain comprising a CD40 messaging domain or a messaging fragment thereof, wherein the cluster domain is capable of oligomerization, thereby activating the messaging domain. The engineered protein of claim 1, wherein the clustering domain is capable of oligomerization by self-assembly. The engineered protein of claim 2, wherein self-assembly comprises the formation of homodimers. The engineered protein of claim 2, wherein self-assembly comprises forming a complex with a receptor. The engineered protein of claim 2, wherein the oligomerization is mediated by ligand binding. The engineered protein of any one of claims 1 to 4, comprising a constitutively stimulating antigen agnostic receptor (C-SAAR). The engineered protein of any one of claims 1 to 4, comprising the C-SAAR of Table 3 or 4. The engineered protein of any one of claims 1 to 4, comprising LZ(cFos)-EGFRTM/JMD-CD28-CD40, LZ(cFos)-CD28TM-CD28-CD40, LZ(cJun)-EGFRTM/ JMD-CD28-CD40, LZ(cJun)-CD28TM-CD28-CD40, LZ(c/EBP)-EGFRTM/JMD-CD28-CD40 or LZ(c/EBP)-CD28TM-CD28-CD40. The engineered protein of any one of claims 1 to 5, comprising an inducible costimulatory receptor. The engineered protein of any one of claims 1 to 5, comprising the inducible costimulatory receptor of Table 5 or 6. The engineered protein of any one of claims 1 to 5, comprising anti-ID1-VH-VL (A30514-pembrolizumab)-CD28TMD-CD28-CD40, or anti-ID1-VL- VH(A30514-pembrolizumab)-CD28TMD-CD28-CD40, or anti-ID2-VH-VL(A30523-pembrolizumab)-CD28TMD-CD28-CD40, or anti-ID2-VL-Vh(A30523- pembrolizumab)-CD28TMD-CD28-CD40, or anti-ID3-Vh-VL(A30633-pembrolizumab)-CD28TMD-CD28-CD40, or anti-ID3-VL-VH(A30633-pembrolizumab) anti)-CD28TMD-CD28-CD40. The engineered protein of claim 1, wherein the engineered protein comprises a transmembrane domain, and the clustering domain oligomerizes when the engineered protein binds to a ligand. The engineered protein of claim 12, wherein the ligand comprises an extracellular ligand. The engineered protein of claim 12, wherein the ligand comprises an intracellular ligand. The engineered protein of claim 1, wherein the engineered protein comprises a transmembrane domain and an extracellular ligand-binding domain, and wherein two or more copies of the engineered protein are produced by ligand The signaling domain is activated when the binding sites remain close to each other. The engineered protein of claim 15, wherein the ligand comprises an antibody. The engineered protein of any one of claims 1 to 14, wherein the engineered protein comprises a clustering domain and a signaling domain comprising a CD40 intracellular domain, wherein when an endogenous T cell receptor (TCR) interacts with When cognate antigens are engaged, the chimeric protein co-stimulates T cells. The engineered protein of claim 17, wherein the clustering domain comprises a T cell receptor (TCR) clustering domain. The engineered protein of claim 17, wherein the clustering domain is activated by extracellular ligand binding. The engineered protein of claim 18, wherein the TCR clustering domain comprises a protein component of the TCR complex or a portion thereof. The engineered protein of claim 18, comprising a T cell receptor integrated antigen indeterminate receptor (TIAAR). The engineered protein of claim 18, comprising a TIAAR from Table 1 or 2. 如請求項18之經工程改造之蛋白質，其包含CD3G_CD3G_CD28CD40及T2A-CD3E_CD3E_CD28CD40、或CD3D_CD3D_CD3D(ICD)_CD28CD40及CD3E_CD3E_CD3E(ICD)_CD28CD40、或CD3D_CD3D_CD3D(ICD)及CD3E_CD3E_CD3E(ICD)、或CD3G_CD3G_CD3G(ICD)_CD28CD40及CD3E_CD3E_CD3E(ICD)_CD28CD40, or CD3G_CD3G_CD3G(ICD) and CD3E_CD3E_CD3E(ICD). The protein of claim 20, wherein the protein of the TCR complex comprises CD3D, CD3E, CD3G, CD3Z, the invariant chain of TCR alpha, the invariant chain of TCR beta, the invariant chain of TCR gamma, or the invariant chain of TCR delta. The protein of claim 20 or 24, wherein the portion thereof comprises a transmembrane portion, an extracellular portion, an intracellular portion, or a combination thereof. The protein of claim 24 or 25, wherein the invariant chain of TCR alpha is co-expressed with the invariant chain of TCR beta. The protein of claim 24 or 25, wherein the invariant chain of TCR gamma is co-expressed with the invariant chain of TCR delta. The protein of any one of claims 1 to 27, wherein the signaling domain further comprises a costimulatory domain. The protein of claim 28, wherein the costimulatory domain is CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40 ), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR) or EphB6 or a portion thereof. The protein of claim 29, wherein the costimulatory domain is CD28. The protein of any one of claims 1 to 30, wherein the protein further comprises a signal peptide. A nucleic acid encoding the protein of any one of claims 1 to 31. A vector comprising the nucleic acid of claim 32. A cell expressing the protein of any one of claims 1 to 31. A cell expressing two or more proteins as claimed in any one of claims 1 to 31. The cell of claim 34 or 35, wherein the cell is an alpha-beta T cell. The cell of claim 36, wherein the alpha-beta T cell is a tumor-infiltrating lymphocyte (TIL). The cell of claim 36 or 37, wherein the TCR clustering domain comprises the invariant chain of TCR gamma co-expressed with the invariant chain of TCR delta. The cell of claim 34 or 35, wherein the cell is a gamma-delta T cell. The cell of claim 39, wherein the TCR clustering domain comprises the invariant chain of TCR alpha co-expressed with the invariant chain of TCR beta. A method of making a cell as claimed in any one of claims 34 to 40, comprising the step of transducing or transfecting the cell with a vector as claimed in claim 33. A method of preparing a cell population expressing the protein of any one of claims 1 to 31, comprising detecting the expression of the protein on the surface of cells transfected or transduced with the vector of claim 33, and selecting Cells were identified as expressing the protein. A cell population produced by the method of claim 42. A cell population enriching cells for expression of the protein of any one of claims 1-31. A method of treating a disease in a subject in need thereof, comprising the step of administering to the subject a cell as claimed in any one of claims 34 to 40 or a population of cells as claimed in claim 43 or 44.

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