HK40061606A

HK40061606A - Chimeric antigen receptors and car-t cells and methods of use

Info

Publication number: HK40061606A
Application number: HK62022049643.9A
Authority: HK
Inventors: A‧佩雷兹; S‧A‧西弗斯; R‧A‧罗德里格斯; J‧贝尔克
Original assignee: 凯德药业股份有限公司
Priority date: 2018-12-12
Filing date: 2019-12-11
Publication date: 2022-06-02

Description

Chimeric antigens and T cell receptors and methods of use

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 62/778,893 filed on 12/2018, which is hereby incorporated by reference in its entirety.

Background

Human cancers are, by their very nature, composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. At this point, the cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These aberrant tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.

Current therapeutic T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct T cells to specific target cancer cells. Chimeric Antigen Receptors (CARs) and engineered T Cell Receptors (TCRs) comprising a binding domain capable of interacting with a specific tumor antigen, allowing T cells to target and kill cancer cells expressing the specific tumor antigen. There is a need for CARs and TCRs for targeting and killing cancer cells.

Summary of The Invention

In at least a first aspect, the present disclosure includes an antigen binding system, antibody or antigen binding fragment thereof comprising an anti-CD 20 binding motif, wherein the anti-CD 20 binding motif comprises the sequence of the three heavy chain complementarity determining regions (HCDRs) of any one Heavy Chain Variable Region (HCVR) selected from the group consisting of SEQ ID NOs 1, 23, 45, 67, 89, 111, 133, 155, 177, and 199, and the sequence of the three light chain cdrs (lcdrs) of any one Light Chain Variable Region (LCVR) selected from the group consisting of SEQ ID NOs 12, 34, 56, 78, 100, 122, 144, 166, 188, and 210. In some embodiments, the anti-CD 20 binding motif comprises a first domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a second domain comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein (i) the HCDR1 has an amino acid sequence according to SEQ ID NOs 3-5, 25-27, 47-49, 69-71, 91-93, 113-; and any one of 201-203; (ii) the HCDR2 has a sequence sum according to any one of SEQ ID NOs 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184 and 204-206; (iii) the HCDR3 has a sequence according to any one of SEQ ID NOs 9-11, 31-33, 53-55, 75-77, 97-99, 119-121, 141-143, 163-165, 185-187 and 207-209; (iv) the LCDR1 has a sequence according to any one of SEQ ID NOs 14-16, 36-38, 58-60, 80-82, 102-104, 124-126, 146-148, 168-170, 190-192 and 212-214; (v) LCDR2 has the sequences shown in SEQ ID NO 17-19, 39-41, 61-63, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and (vi) the LCDR3 has a sequence according to any one of SEQ ID NOs 20-22, 42-44, 64-66, 86-88, 108-. In some embodiments, the HCDR comprises: (i) HCDR1 according to any one of SEQ ID NOs 3-5; HCDR2 according to any one of SEQ ID NOs 6-8; HCDR3 according to any one of SEQ ID NOs 9-11; (ii) HCDR1 according to any one of SEQ ID NOs 25-27; HCDR2 according to any one of SEQ ID NOs 28-30; HCDR3 according to any one of SEQ ID NOs 31-33; (iii) HCDR1 according to any one of SEQ ID NOs 47-49; HCDR2 according to any one of SEQ ID NOs 50-52; HCDR3 according to any one of SEQ ID NOs 53-55; (iv) HCDR1 according to any one of SEQ ID NOs 69-71; HCDR2 according to any one of SEQ ID NOs 72-74; HCDR3 according to any one of SEQ ID NOs 75-77; (v) HCDR1 according to any one of SEQ ID NOs 91-93; HCDR2 according to any one of SEQ ID NOs 94-96; HCDR3 according to any one of SEQ ID NOs 97-99; (vi) HCDR1 according to any one of SEQ ID NO 113-115; HCDR2 according to any one of SEQ ID NO: 116-118; HCDR3 according to any one of SEQ ID NO 119-121; (vii) HCDR1 according to any one of SEQ ID NO 135-137; HCDR2 according to any one of SEQ ID NO 138-140; HCDR3 according to any one of SEQ ID NO 141-143; (viii) HCDR1 according to any one of SEQ ID NO 157-159; HCDR2 according to any one of SEQ ID NOs 160-162; HCDR3 according to any one of SEQ ID NO 163-165; (ix) HCDR1 according to any one of SEQ ID NO 179-181; HCDR2 according to any one of SEQ ID NO 182-184; HCDR3 according to any one of SEQ ID NO 185-187; or (x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO 204-206; HCDR3 according to any one of SEQ ID NO 207-209; and the LCDR comprises: (i) LCDR1 according to any one of SEQ ID NOs 14-16; LCDR2 according to any one of SEQ ID NOs 17-19; LCDR3 according to any one of SEQ ID NOs 20-22; (ii) LCDR1 according to any one of SEQ ID NOs 36-38; LCDR2 according to any one of SEQ ID NOs 39-41; LCDR3 according to any one of SEQ ID NOS 42-44; (iii) LCDR1 according to any one of SEQ ID NOs 58-60; LCDR2 according to any one of SEQ ID NOs 61-63; LCDR3 according to any one of SEQ ID NOs 64-66; (iv) LCDR1 according to any one of SEQ ID NOs 80-82; LCDR2 according to any one of SEQ ID NOs 83-85; LCDR3 according to any one of SEQ ID NOs 86-88; (v) LCDR1 according to any one of SEQ ID NO 102-104; LCDR2 according to any one of SEQ ID NO 105-107; LCDR3 according to any one of SEQ ID NO 108-110; (vi) LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO 127-129; LCDR3 of any one of SEQ ID NO: 130-132; (vii) LCDR1 according to any one of SEQ ID NO 146-148; LCDR2 according to any one of SEQ ID NO 149-151; LCDR3 according to any one of SEQ ID NO 152-154; (viii) LCDR1 according to any one of SEQ ID NO 168-170; LCDR2 according to any one of SEQ ID NO 171-173; LCDR3 according to any one of SEQ ID NO: 174-176; (ix) LCDR1 according to any one of SEQ ID NO 190-192; LCDR2 according to any one of SEQ ID NO 193-195; LCDR3 according to any one of SEQ ID NO 196-198; or (x) LCDR1 according to any one of SEQ ID NO:212 and 214; LCDR2 according to any of SEQ ID NO:215 and 217; LCDR3 according to any one of SEQ ID NO 218-220.

In various embodiments, the antigen binding system, antibody or antigen binding fragment thereof of the present disclosure comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), wherein: the HCDR and LCDR comprise: (i) HCDR1 according to any one of SEQ ID NOs 3-5; HCDR2 according to any one of SEQ ID NOs 6-8; HCDR3 according to any one of SEQ ID NOs 9-11; LCDR1 according to any one of SEQ ID NOs 14-16; LCDR2 according to any one of SEQ ID NOs 17-19; LCDR3 according to any one of SEQ ID NOs 20-22; (ii) HCDR1 according to any one of SEQ ID NOs 25-27; HCDR2 according to any one of SEQ ID NOs 28-30; HCDR3 according to any one of SEQ ID NOs 31-33; LCDR1 according to any one of SEQ ID NOs 36-38; LCDR2 according to any one of SEQ ID NOs 39-41; LCDR3 according to any one of SEQ ID NOS 42-44; (iii) HCDR1 according to any one of SEQ ID NOs 47-49; HCDR2 according to any one of SEQ ID NOs 50-52; HCDR3 according to any one of SEQ ID NOs 53-55; LCDR1 according to any one of SEQ ID NOs 58-60; LCDR2 according to any one of SEQ ID NOs 61-63; LCDR3 according to any one of SEQ ID NOs 64-66; (iv) HCDR1 according to any one of SEQ ID NOs 69-71; HCDR2 according to any one of SEQ ID NOs 72-74; HCDR3 according to any one of SEQ ID NOs 75-77; LCDR1 according to any one of SEQ ID NOs 80-82; LCDR2 according to any one of SEQ ID NOs 83-85; LCDR3 according to any one of SEQ ID NOs 86-88; (v) HCDR1 according to any one of SEQ ID NOs 91-93; HCDR2 according to any one of SEQ ID NOs 94-96; HCDR3 according to any one of SEQ ID NOs 97-99; LCDR1 according to any one of SEQ ID NO 102-104; LCDR2 according to any one of SEQ ID NO 105-107; LCDR3 according to any one of SEQ ID NO 108-110; (vi) HCDR1 according to any one of SEQ ID NO 113-115; HCDR2 according to any one of SEQ ID NO: 116-118; HCDR3 according to any one of SEQ ID NO 119-121; LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO 127-129; LCDR3 of any one of SEQ ID NO: 130-132; (vii) HCDR1 according to any one of SEQ ID NO 135-137; HCDR2 according to any one of SEQ ID NO 138-140; HCDR3 according to any one of SEQ ID NO 141-143; LCDR1 according to any one of SEQ ID NO 146-148; LCDR2 according to any one of SEQ ID NO 149-151; LCDR3 according to any one of SEQ ID NO 152-154; (viii) HCDR1 according to any one of SEQ ID NO 157-159; HCDR2 according to any one of SEQ ID NOs 160-162; HCDR3 according to any one of SEQ ID NO 163-165; LCDR1 according to any one of SEQ ID NO 168-170; LCDR2 according to any one of SEQ ID NO 171-173; LCDR3 according to any one of SEQ ID NO: 174-176; (ix) HCDR1 according to any one of SEQ ID NO 179-181; HCDR2 according to any one of SEQ ID NO 182-184; HCDR3 according to any one of SEQ ID NO 185-187; LCDR1 according to any one of SEQ ID NO 190-192; LCDR2 according to any one of SEQ ID NO 193-195; LCDR3 according to any one of SEQ ID NO 196-198; or (x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO 204-206; HCDR3 according to any one of SEQ ID NO 207-209; LCDR1 according to any one of SEQ ID NO 212-214; LCDR2 according to any of SEQ ID NO:215 and 217; LCDR3 according to any one of SEQ ID NO 218-220. In some embodiments, the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO 1, 23, 45, 67, 89, 111, 133, 155, 177 or 199; and (ii) the light chain variable domain is at least 80% identical to SEQ ID NO 12, 34, 56, 78, 100, 122, 144, 166, 188 or 210. In some embodiments, the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO 1 and the light chain variable domain is at least 80% identical to SEQ ID NO 12; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 23 and the light chain variable domain is at least 80% identical to SEQ ID NO 34; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 45 and the light chain variable domain is at least 80% identical to SEQ ID NO 56; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO 67 and the light chain variable domain is at least 80% identical to SEQ ID NO 78; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO 89 and the light chain variable domain is at least 80% identical to SEQ ID NO 100; (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO 111 and the light chain variable domain is at least 80% identical to SEQ ID NO 122; (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 133 and the light chain variable domain is at least 80% identical to SEQ ID NO 144; (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 155 and the light chain variable domain is at least 80% identical to SEQ ID NO 166; (ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO:177 and the light chain variable domain is at least 80% identical to SEQ ID NO: 188; or (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO:199 and the light chain variable domain is at least 80% identical to SEQ ID NO: 210.

In some embodiments of the disclosure comprising three HCDRs and three LCDRs, the three HCDRs and the three LCDRs are comprised by a single polypeptide. In some embodiments of the disclosure comprising three HCDRs and three LCDRs, the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide. In some embodiments, the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain.

In some embodiments, the antigen binding system, antibody or antigen binding fragment thereof further comprises: (i) a binding motif that binds to an antigen selected from the group consisting of: 5T4, alpha-fetoprotein, B-cell maturation antigen (BCMA), B-cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, C-Met, CSPG4, IgM, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT 2, folate binding protein, GD2, HER2-HER 2 combination, HER2/Neu, HERV-2-1 envelope glycoprotein gp 2, HIV-1 envelope glycoprotein gpl2, IL-rall 2, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p 2, mutated ras, VEGFR 72, VEGFR-specific receptor (ephfr 2), ephha 2), and ephha receptor 2 (ephha 2); and/or (ii) a binding motif that binds to an antigen characteristic of a B cell, optionally wherein the antigen characteristic of a B cell is not CD19 or CD 20. In some embodiments, the antigen binding system, antibody, or antigen binding fragment thereof further comprises an anti-CD 19 binding motif. In some embodiments, the anti-CD 19 binding motif comprises a first domain comprising three HCDRs and a second domain comprising three LCDRs, wherein: the three HCDRs of the anti-CD 19 binding motif comprise HCDR1, HCDR2, and HCDR 3; the three LCDRs of the anti-CD 19 binding motif comprise LCDR1, LCDR2, and LCDR 3; and the HCDR and LCDR of the anti-CD 19 binding motif comprise HCDR1 according to any one of SEQ ID NO: 223-225; HCDR2 according to any one of SEQ ID NO 226-228; HCDR3 according to any one of SEQ ID NO 229-231; LCDR1 according to any one of SEQ ID NO 234-236; LCDR2 according to any one of SEQ ID NO 237 and 239; LCDR3 according to any one of SEQ ID NO 240-242. In some embodiments, the anti-CD 19 binding motif comprises a first heavy chain variable domain comprising three HCDRs of the anti-CD 19 binding motif and a light chain variable domain comprising three LCDRs of the anti-CD 19 binding motif, wherein the heavy chain variable domain of the anti-CD 19 binding motif is at least 80% identical to SEQ ID No. 221 and the light chain variable domain of the anti-CD 19 binding motif is at least 80% identical to SEQ ID No. 232. In some embodiments, the three HCDRs of the anti-CD 19 binding motif and the three LCDRs of the anti-CD 19 binding motif are comprised by a single polypeptide. In some embodiments, the three HCDRs of the anti-CD 20 binding motif, the three LCDRs of the anti-CD 20 binding motif, the three HCDRs of the anti-CD 19 binding motif, and the three LCDRs of the anti-CD 19 binding motif together are comprised by a single polypeptide.

In various embodiments, the antigen binding system, antibody, or antigen binding fragment thereof is or is comprised by a chimeric antigen receptor. In some embodiments, the antigen binding system, antibody, or antigen binding fragment thereof is a single polypeptide that is or is comprised by a chimeric antigen receptor that is a bispecific chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain, i.e., the transmembrane domain of 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of a T cell receptor, or any combination thereof. In some embodiments, (i) the three HCDRs of the anti-CD 20 binding motif and the three LCDRs of the anti-CD 20 binding motif are present in a first polypeptide, and (ii) the three HCDRs of the anti-CD 19 binding motif and the three LCDRs of the anti-CD 19 binding motif together are comprised by a second, different polypeptide. In some embodiments, the first polypeptide is or is comprised by a first chimeric antigen receptor. In some embodiments, the second polypeptide is or is comprised by a second chimeric antigen receptor.

In various embodiments, the present disclosure comprises nucleic acids encoding at least one polypeptide of the disclosure and/or vectors comprising such nucleic acids. The disclosure further comprises a method of producing an engineered cell comprising transfecting or transducing a cell with a nucleic acid encoding at least one polypeptide of the disclosure. Further provided herein are cells encoding or expressing an antigen binding system, antibody or antigen binding fragment provided herein, optionally wherein the cells are immune cells, optionally wherein the cells are T cells.

The present disclosure further comprises a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising an antigen binding system, antibody, or antigen binding fragment thereof of the present disclosure. Also provided herein are methods of inducing an immune response in a subject or immunizing a subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising an antigen binding system, antibody, or antigen binding fragment thereof of the present disclosure. In some embodiments, the cell is a CAR-T cell. In various embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), hairy cell leukemia, hodgkin's disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, Monoclonal Gammopathy of Unknown Significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin's lymphoma (NHL), Plasma cell proliferative disorders (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmacytoma, plasmacytoid dendritic cell neoplasms, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small-cell or large-cell follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformative follicular lymphoma or Walsh macroglobulinemia, Mantle Cell Lymphoma (MCL), Transformative Follicular Lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), Multiple myeloma, hairy cell lymphoma/leukemia or a combination thereof. In some embodiments, the cell therapy is allogeneic or autologous cell therapy.

At least one aspect of the present disclosure includes a chimeric antigen receptor comprising an anti-CD 20 binding motif, the anti-CD 20 binding motif comprising three heavy chain complementarity determining regions (HCDRs) and three light chain cdrs (LCDRs), the three HCDRs being contained within a Heavy Chain Variable Region (HCVR) sequence selected from the group consisting of SEQ ID NOs 1, 23, 45, 67, 89, 111, 133, 155, 177, and 199, and the three LCDRs being contained within a Heavy Chain Variable Region (HCVR) sequence selected from the group consisting of SEQ ID NOs 12, 34, 56, 78, 100, 122, 144, 166, 188, and 210. In some embodiments, the first domain comprises three heavy chain complementarity determining regions (HCDRs) and the second domain comprises three light chain complementarity determining regions (LCDRs), wherein (i) the HCDR1 has a sequence according to SEQ ID NOS 3-5, 25-27, 47-49, 69-71, 91-93, 113-; and any one of 201-203; (ii) the HCDR2 has the sequences shown in SEQ ID NOs 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184; and 204-206; (iii) the HCDR3 has the sequences in accordance with SEQ ID NO 9-11, 31-33, 53-55, 75-77, 97-99, 119-121, 141-143, 163-165, 185-187; and 207-209; (iv) the LCDR1 has the sequences shown in SEQ ID NO 14-16, 36-38, 58-60, 80-82, 102, 124, 146, 148, 168, 170, 190, 192; and 212-214; (v) LCDR2 has the sequences shown in SEQ ID NO 17-19, 39-41, 61-63, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and (vi) the LCDR3 has the sequence shown in SEQ ID NO:20-22, 42-44, 64-66, 86-88, 108-; and any of 218 and 220. In some embodiments, the HCDR comprises: (i) HCDR1 according to any one of SEQ ID NOs 3-5; HCDR2 according to any one of SEQ ID NOs 6-8; HCDR3 according to any one of SEQ ID NOs 9-11; (ii) HCDR1 according to any one of SEQ ID NOs 25-27; HCDR2 according to any one of SEQ ID NOs 28-30; HCDR3 according to any one of SEQ ID NOs 31-33; (iii) HCDR1 according to any one of SEQ ID NOs 47-49; HCDR2 according to any one of SEQ ID NOs 50-52; HCDR3 according to any one of SEQ ID NOs 53-55; (iv) HCDR1 according to any one of SEQ ID NOs 69-71; HCDR2 according to any one of SEQ ID NOs 72-74; HCDR3 according to any one of SEQ ID NOs 75-77; (v) HCDR1 according to any one of SEQ ID NOs 91-93; HCDR2 according to any one of SEQ ID NOs 94-96; HCDR3 according to any one of SEQ ID NOs 97-99; (vi) HCDR1 according to any one of SEQ ID NO 113-115; HCDR2 according to any one of SEQ ID NO: 116-118; HCDR3 according to any one of SEQ ID NO 119-121; (vii) HCDR1 according to any one of SEQ ID NO 135-137; HCDR2 according to any one of SEQ ID NO 138-140; HCDR3 according to any one of SEQ ID NO 141-143; (viii) HCDR1 according to any one of SEQ ID NO 157-159; HCDR2 according to any one of SEQ ID NOs 160-162; HCDR3 according to any one of SEQ ID NO 163-165; (ix) HCDR1 according to any one of SEQ ID NO 179-181; HCDR2 according to any one of SEQ ID NO 182-184; HCDR3 according to any one of SEQ ID NO 185-187; or (x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO 204-206; HCDR3 according to any one of SEQ ID NO 207-209; and the LCDR comprises: (i) LCDR1 according to any one of SEQ ID NOs 14-16; LCDR2 according to any one of SEQ ID NOs 17-19; LCDR3 according to any one of SEQ ID NOs 20-22; (ii) LCDR1 according to any one of SEQ ID NOs 36-38; LCDR2 according to any one of SEQ ID NOs 39-41; LCDR3 according to any one of SEQ ID NOS 42-44; (iii) LCDR1 according to any one of SEQ ID NOs 58-60; LCDR2 according to any one of SEQ ID NOs 61-63; LCDR3 according to any one of SEQ ID NOs 64-66; (iv) LCDR1 according to any one of SEQ ID NOs 80-82; LCDR2 according to any one of SEQ ID NOs 83-85; LCDR3 according to any one of SEQ ID NOs 86-88; (v) LCDR1 according to any one of SEQ ID NO 102-104; LCDR2 according to any one of SEQ ID NO 105-107; LCDR3 according to any one of SEQ ID NO 108-110; (vi) LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO 127-129; LCDR3 according to any one of SEQ ID NO: 130-132; (vii) LCDR1 according to any one of SEQ ID NO 146-148; LCDR2 according to any one of SEQ ID NO 149-151; LCDR3 according to any one of SEQ ID NO 152-154; (viii) LCDR1 according to any one of SEQ ID NO 168-170; LCDR2 according to any one of SEQ ID NO 171-173; LCDR3 according to any one of SEQ ID NO: 174-176; (ix) LCDR1 according to any one of SEQ ID NO 190-192; LCDR2 according to any one of SEQ ID NO 193-195; LCDR3 according to any one of SEQ ID NO 196-198; or (x) LCDR1 according to any one of SEQ ID NO:212 and 214; LCDR2 according to any of SEQ ID NO:215 and 217; LCDR3 according to any one of SEQ ID NO 218-220.

In some embodiments, the chimeric antigen receptor comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), wherein: the three HCDRs include HCDR1, HCDR2, and HCDR 3; the three LCDRs include LCDR1, LCDR2, and LCDR 3; and the HCDR and LCDR include: (i) HCDR1 according to any one of SEQ ID NOs 3-5; HCDR2 according to any one of SEQ ID NOs 6-8; HCDR3 according to any one of SEQ ID NOs 9-11; LCDR1 according to any one of SEQ ID NOs 14-16; LCDR2 according to any one of SEQ ID NOs 17-19; LCDR3 according to any one of SEQ ID NOs 20-22; (ii) HCDR1 according to any one of SEQ ID NOs 25-27; HCDR2 according to any one of SEQ ID NOs 28-30; HCDR3 according to any one of SEQ ID NOs 31-33; LCDR1 according to any one of SEQ ID NOs 36-38; LCDR2 according to any one of SEQ ID NOs 39-41; LCDR3 according to any one of SEQ ID NOS 42-44; (iii) HCDR1 according to any one of SEQ ID NOs 47-49; HCDR2 according to any one of SEQ ID NOs 50-52; HCDR3 according to any one of SEQ ID NOs 53-55; LCDR1 according to any one of SEQ ID NOs 58-60; LCDR2 according to any one of SEQ ID NOs 61-63; LCDR3 according to any one of SEQ ID NOs 64-66; (iv) HCDR1 according to any one of SEQ ID NOs 69-71; HCDR2 according to any one of SEQ ID NOs 72-74; HCDR3 according to any one of SEQ ID NOs 75-77; LCDR1 according to any one of SEQ ID NOs 80-82; LCDR2 according to any one of SEQ ID NOs 83-85; LCDR3 according to any one of SEQ ID NOs 86-88; (v) HCDR1 according to any one of SEQ ID NOs 91-93; HCDR2 according to any one of SEQ ID NOs 94-96; HCDR3 according to any one of SEQ ID NOs 97-99; LCDR1 according to any one of SEQ ID NO 102-104; LCDR2 according to any one of SEQ ID NO 105-107; LCDR3 according to any one of SEQ ID NO 108-110; (vi) HCDR1 according to any one of SEQ ID NO 113-115; HCDR2 according to any one of SEQ ID NO: 116-118; HCDR3 according to any one of SEQ ID NO 119-121; LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO 127-129; LCDR3 according to any one of SEQ ID NO: 130-132; (vii) HCDR1 according to any one of SEQ ID NO 135-137; HCDR2 according to any one of SEQ ID NO 138-140; HCDR3 according to any one of SEQ ID NO 141-143; LCDR1 according to any one of SEQ ID NO 146-148; LCDR2 according to any one of SEQ ID NO 149-151; LCDR3 according to any one of SEQ ID NO 152-154; (viii) HCDR1 according to any one of SEQ ID NO 157-159; HCDR2 according to any one of SEQ ID NOs 160-162; HCDR3 according to any one of SEQ ID NO 163-165; LCDR1 according to any one of SEQ ID NO 168-170; LCDR2 according to any one of SEQ ID NO 171-173; LCDR3 according to any one of SEQ ID NO: 174-176; (ix) HCDR1 according to any one of SEQ ID NO 179-181; HCDR2 according to any one of SEQ ID NO 182-184; HCDR3 according to any one of SEQ ID NO 185-187; LCDR1 according to any one of SEQ ID NO 190-192; LCDR2 according to any one of SEQ ID NO 193-195; LCDR3 according to any one of SEQ ID NO 196-198; or (x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO 204-206; HCDR3 according to any one of SEQ ID NO 207-209; LCDR1 according to any one of SEQ ID NO 212-214; LCDR2 according to any of SEQ ID NO:215 and 217; LCDR3 according to any one of SEQ ID NO 218-220. In various embodiments, the chimeric antigen receptor comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO 1, 23, 45, 67, 89, 111, 133, 155, 177 or 199; and (ii) the light chain variable domain is at least 80% identical to SEQ ID NO 12, 34, 56, 78, 100, 122, 144, 166, 188 or 210. In some embodiments, the chimeric antigen receptor comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO 1 and the light chain variable domain is at least 80% identical to SEQ ID NO 12; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 23 and the light chain variable domain is at least 80% identical to SEQ ID NO 34; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 45 and the light chain variable domain is at least 80% identical to SEQ ID NO 56; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO 67 and the light chain variable domain is at least 80% identical to SEQ ID NO 78; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO. 89 and the light chain variable domain is at least 80% identical to SEQ ID NO. 100; (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO 111 and the light chain variable domain is at least 80% identical to SEQ ID NO 122; (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 133 and the light chain variable domain is at least 80% identical to SEQ ID NO 144; (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 155 and the light chain variable domain is at least 80% identical to SEQ ID NO 166; (ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO:177 and the light chain variable domain is at least 80% identical to SEQ ID NO: 188; or (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO:199 and the light chain variable domain is at least 80% identical to SEQ ID NO: 210.

In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs and the three LCDRs are comprised by a single polypeptide. In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide. In some embodiments, the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain. In some embodiments, the chimeric antigen receptor further comprises: (i) a binding motif that specifically binds to an antigen selected from the group consisting of: 5T4, alpha-fetoprotein, B-cell maturation antigen (BCMA), B-cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, C-Met, CSPG4, IgM, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT 2, folate binding protein, GD2, HER2-HER 2 combination, HER2/Neu, HERV-2-1 envelope glycoprotein gp 2, HIV-1 envelope glycoprotein gpl2, IL-rall 2, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p 2, mutated ras, VEGFR 72, VEGFR-specific receptor (ephfr 2), ephha 2), and ephha receptor 2 (ephha 2); and/or (ii) a binding motif that specifically binds to a B cell characteristic antigen, optionally wherein the B cell characteristic antigen is not CD19 or CD 20. In some embodiments, the chimeric antigen receptor further comprises an anti-CD 19 binding motif.

In some embodiments, the chimeric antigen receptor of claim 40, wherein the anti-CD 19 binding motif comprises a first domain comprising three HCDRs and a second domain comprising three LCDRs, wherein: the three HCDRs of the anti-CD 19 binding motif comprise HCDR1, HCDR2, and HCDR 3; the three LCDRs of the anti-CD 19 binding motif comprise LCDR1, LCDR2, and LCDR 3; and the HCDR and LCDR of the anti-CD 19 binding motif comprise HCDR1 according to any one of SEQ ID NO: 223-225; HCDR2 according to any one of SEQ ID NO 226-228; HCDR3 according to any one of SEQ ID NO 229-231; LCDR1 according to any one of SEQ ID NO 234-236; LCDR2 according to any one of SEQ ID NO 237 and 239; LCDR3 according to any one of SEQ ID NO 240-242. In some embodiments, the anti-CD 19 binding motif comprises a first heavy chain variable domain comprising three HCDRs of the anti-CD 19 binding motif and a light chain variable domain comprising three LCDRs of the anti-CD 19 binding motif, wherein the heavy chain variable domain of the anti-CD 19 binding motif is at least 80% identical to SEQ ID No. 221 and the light chain variable domain of the anti-CD 19 binding motif is at least 80% identical to SEQ ID No. 232. In some embodiments, the three HCDRs of the anti-CD 19 binding motif and the three LCDRs of the anti-CD 19 binding motif are comprised by a single polypeptide.

In some embodiments, the three HCDRs of the anti-CD 20 binding motif, the three LCDRs of the anti-CD 20 binding motif, the three HCDRs of the anti-CD 19 binding motif, and the three LCDR motifs of the anti-CD 19 binding motif together are comprised by a single polypeptide. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that is the transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.

In various embodiments, the present disclosure comprises a bicistronic chimeric antigen receptor comprising a first chimeric antigen receptor of the present disclosure and a second chimeric antigen receptor comprising a binding motif that specifically binds to an antigen selected from the group consisting of: 5T4, alpha-fetoprotein, B-cell maturation antigen (BCMA), B-cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, C-Met, CSPG4, IgM, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT 2, folate binding protein, GD2, HER2-HER 2 combination, HER2/Neu, HERV-2-1 envelope glycoprotein gp 2, HIV-1 envelope glycoprotein gpl2, IL-rall 2, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p 2, mutated ras, VEGFR 72, VEGFR-specific receptor (ephfr 2), ephha 2), and ephha receptor 2 (ephha 2); and/or a binding motif that specifically binds to a B cell characteristic antigen, optionally wherein the B cell characteristic antigen is not CD19 or CD 20. In some embodiments, the second chimeric antigen receptor comprises an anti-CD 19 binding motif.

The present disclosure further provides nucleic acids encoding at least one polypeptide of the disclosure and/or vectors comprising such nucleic acids. The disclosure also encompasses methods of generating an engineered cell comprising transfecting or transducing a cell with a nucleic acid of the disclosure. In various embodiments, the present disclosure comprises a cell encoding or expressing a chimeric antigen receptor provided herein, optionally wherein the cell is an immune cell, optionally wherein the cell is a T cell.

The present disclosure further provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising a chimeric antigen receptor of the present disclosure. Further provided herein are methods of inducing an immune response in a subject or immunizing a subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising a chimeric antigen receptor of the present disclosure. In various embodiments, the cell is a CAR-T cell. In various embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), hairy cell leukemia, hodgkin's disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, Monoclonal Gammopathy of Unknown Significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin's lymphoma (NHL), Plasma cell proliferative disorders (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmacytoma, plasmacytoid dendritic cell neoplasms, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small-cell or large-cell follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformative follicular lymphoma or Walsh macroglobulinemia, Mantle Cell Lymphoma (MCL), Transformative Follicular Lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), Multiple myeloma, hairy cell lymphoma/leukemia or a combination thereof. In some embodiments, the cell therapy is allogeneic or autologous cell therapy.

In at least one aspect, the present disclosure includes a chimeric antigen receptor comprising an anti-CD 20 binding motif and a CD19 binding motif, wherein the anti-CD 20 binding motif comprises an amino acid sequence selected from the group consisting of: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210. In some embodiments, the CD19 binding motif comprises an amino acid sequence selected from the group consisting of: 221 and 232, SEQ ID NO. In some embodiments, the anti-CD 20 binding motif comprises an amino acid sequence selected from the group consisting of: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210; wherein the CD19 binding motif comprises an amino acid sequence selected from the group consisting of: 221 and 232, SEQ ID NO. In some embodiments, the anti-CD 20 binding motif and the CD19 binding motif are comprised by a single polypeptide. In some embodiments, the anti-CD 20 binding motif and the CD19 binding motif are comprised by different polypeptides.

In at least one aspect, the present disclosure comprises a polynucleotide encoding an amino acid sequence selected from the group consisting of: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210. In at least one aspect, the present disclosure includes a pharmaceutical composition comprising a chimeric antigen receptor comprising an anti-CD 20 binding motif having an amino acid sequence selected from the group consisting of seq id nos: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210. In some embodiments, the composition further comprises a CD19 binding motif.

Detailed Description

The present disclosure relates to novel polypeptides comprising novel antigen binding molecules and polynucleotides encoding the same. Some aspects of the present disclosure relate to polynucleotides encoding a Chimeric Antigen Receptor (CAR) comprising at least one of the heavy and light chains (or CDRs thereof) disclosed herein. The disclosure also provides vectors (e.g., viral vectors) comprising such polynucleotides and compositions comprising such vectors. The disclosure further provides polynucleotides encoding such CARs or TCRs and compositions comprising such polynucleotides. The present disclosure additionally provides engineered cells (e.g., T cells) comprising such polynucleotides and/or transduced with such viral vectors, and compositions comprising such engineered cells. The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising a plurality of engineered T cells. The present disclosure provides methods for making such engineered T cells and compositions and uses of such engineered T cells and compositions (e.g., for treating melanoma). Also, the present disclosure provides a method of inducing immunity against a tumor comprising administering to a subject an effective amount of a cell comprising a polynucleotide, vector or polypeptide of the present disclosure. Other aspects of the disclosure relate to cells comprising a CAR and their use in T cell therapy for treating a patient having cancer.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. While the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference. Other features and advantages of the disclosure will be apparent from the following detailed description, including the embodiments and the claims.

Definition of

In order that the disclosure of the invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" should be understood to be inclusive and encompass both "or" and "unless the context clearly dictates otherwise.

The term "and/or" as used herein is to be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" is intended to include a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The terms "for example (e.g.)" and "i.e. (i.e.)" as used herein are used by way of example only and are not intended to be limiting and should not be construed to relate only to those items specifically enumerated in the specification.

The term "or more", "at least", "over", etc., e.g., "at least one" should be understood to include, but not be limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 100, 105, 104, 102, 105, 108, 102, 109, 106, 105, 108, 109, 106, 109, 100, 105, 106, 105, 109, 103, 45, 47, 48, 49, 50, 60, 62, 63, 60, 62, 63, 64, 65, 67, 60, 6, 60, 6, and so on, 110. 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated values. But also any larger number or fraction therebetween.

Conversely, the term "not more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. But also any smaller number or fraction therebetween.

The terms "plurality", "at least two", "two or more", "at least a second", etc. should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 104, 107, 106, 105, 106, 107, 102, 106, 105, 106, 105, 45, 25, 48, 49, 27, 60, 61, 62, 25, 60, 62, 65, 67, 60, 67, 60, 6, 60, 25, 61, 25, 60, 25, 60, 65, 60, 65, 6, 65, 60, 6, 60, 65, 6, 60, 6, 25, 60, 25, 60, 25, 65, 60, 25, and so on, 109. 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. But also any larger number or fraction therebetween.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It should be understood that the language "comprising" is used herein to describe aspects and also to provide other similar aspects described as "consisting of and/or" consisting essentially of.

Unless specifically stated or otherwise apparent from the context, the term "about," as used herein, refers to a value or composition within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of may mean within 1 or more than 1 standard deviation as practiced in the art. "about" or "consisting essentially of may represent a range of up to 10% (i.e., ± 10%). Thus, "about" may be understood as being greater than or less than the stated value within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001%. For example, about 5mg may include any amount between 4.5mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may represent values up to an order of magnitude or up to 5-fold. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "consisting essentially of … …" should be assumed to be within an acceptable error range for that particular value or composition.

As used herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range is to be understood as encompassing the value of any integer within the recited range, and where appropriate, including fractions thereof (e.g., tenths and hundredths of integers).

The units, prefixes, and symbols used herein are provided in their international system of units (SI) accepted form. Numerical ranges include the numbers defining the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Juo, "The circumcise Dictionary of Biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5th ed., (2013), Academic Press; and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammacack et al.

By "administering" is meant physically introducing the agent into the subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection and including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, such as orally. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration may also be carried out, for example, once, multiple times, and/or over one or more extended periods.

The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the Ab may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). Typically, human antibodies are tetrameric agents of approximately 150kD, consisting of two identical heavy (H) chain polypeptides (each about 50kD) and two identical light (L) chain polypeptides (each about 25kD), which associate with each other into what is commonly referred to as a "Y-shaped" structure. The heavy and light chains are interconnected (link) or joined (connect) by a single disulfide bond; two additional disulfide bonds link the heavy chain hinge regions to each other, thereby linking the dimers to each other and forming tetramers. Naturally occurring antibodies are also glycosylated, for example, on the CH2 domain.

The term "human antibody" is intended to encompassAn antibody having variable domain and constant domain sequences generated, assembled or derived from human immunoglobulin sequences, or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) can be considered "human", even if their amino acid sequences comprise residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., variations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "humanized" is intended to encompass antibodies having variable domains with sequences derived from those of a non-human species (e.g., mouse) modified to more closely resemble the sequences encoded by a human germline. In some embodiments, a "humanized" antibody comprises one or more framework domains having substantially the amino acid sequence of a human framework domain, and one or more complementarity determining regions having substantially the same amino acid sequence as that of a non-human antibody. In some embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant domain. In some embodiments, the humanized antibody may comprise a C of a human heavy chain constant domain _H1. Hinge, C_H2、C_H3 and optionally C_H4。

Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain fv (scfv), camelized antibodies, affibodies, Fab fragments, F (ab')₂Fragments, disulfide-linked fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "Antibody mimetics ") and antigen binding fragments of any of the above. In certain embodiments, an antibody described herein refers to a polyclonal antibody population. The antibody may also comprise, for example, a Fab' fragment, Fd fragment, isolated CDR, single chain Fv, polypeptide-Fc fusion, single domain antibody (e.g., a shark single domain antibody, such as IgNAR or a fragment thereof), camelid antibody, single chain or tandem diabody A minibody,Ankyrin repeat proteins orDART, TCR-like antibody,MicroProteins、And

the immunoglobulin may be derived from any well-known isotype, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM. The IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG 4. "isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, for example, both naturally occurring and non-naturally occurring abs; monoclonal and polyclonal Ab; chimeric and humanized abs; human or non-human Ab; fully synthetic Ab; and a single chain Ab. Non-human abs may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or antigen-binding portions of any of the foregoing immunoglobulins, and includes monovalent and divalent fragments, as well as single chain abs.

An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that comprises an antigen-binding portion (e.g., a CDR) from an antibody from which the molecule is derived. The antigen binding molecule may include antigen Complementarity Determining Regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments, dabs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide binding domain) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or binds to a viral or bacterial antigen. In certain embodiments, the antigen binding molecule binds to BCMA, CLL-1, or FLT 3. In certain embodiments, the antigen binding molecule binds to CD19, CD20, or both. In a further embodiment, the antigen binding molecule is an antibody fragment that specifically binds an antigen, including one or more Complementarity Determining Regions (CDRs) thereof. In a further embodiment, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of a high affinity multimer (avimer).

In some cases, the CDRs are substantially identical to the sequences of the CDRs found in a reference antibody (e.g., an antibody of the disclosure) and/or the CDRs provided in the disclosure. In some embodiments, a CDR is substantially identical to a reference CDR (e.g., a CDR provided in the present disclosure) in that it is identical in sequence or contains between 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions as compared to the reference CDR. In some embodiments, a CDR is substantially identical to a reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that one amino acid is deleted, added, or substituted within the CDR as compared to the reference CDR, while the CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference. In some embodiments, the CDR is substantially identical to the reference CDR in that 2, 3, 4, or 5 (e.g., 2-5) amino acids within the CDR are deleted, added, or substituted as compared to the reference CDR, while the CDR has an amino acid sequence that is otherwise identical to the reference CDR. In various embodiments, the antigen-binding fragment binds to the same antigen as the reference antibody.

Antigen binding fragments can be produced by any means. For example, in some embodiments, an antigen-binding fragment may be enzymatically or chemically produced by fragmentation of an intact antibody. In some embodiments, the antigen-binding fragment may be produced recombinantly (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, the antigen-binding fragment may be produced synthetically, in whole or in part. In some embodiments, an antigen-binding fragment can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more; in some embodiments at least about 200 amino acids (e.g., 50-100, 50-150, 50-200, or 100-200 amino acids).

As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region generally refers to a portion of an antibody, typically a light chain or a portion of a heavy chain, typically about the amino terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ greatly in sequence between antibodies and are used for binding and specificity of a particular antibody for its particular antigen. The variability of the sequence is concentrated in those regions called Complementarity Determining Regions (CDRs), while the more highly conserved regions in the variable domains are called Framework Regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises a rodent or murine CDR and a human Framework Region (FR). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises a rodent or murine CDR and a primate (e.g., non-human primate) Framework Region (FR).

The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen binding molecule thereof.

The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen binding molecule thereof.

Some definitions of CDRs are commonly used: kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. contact definition is based on analysis of available complex crystal structures.

Table 1: CDR numbering

The term "Kabat numbering" and similar terms are art-recognized and refer to the numbering system of amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding molecule thereof. In certain aspects, the CDRs of an antibody may be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382 + 391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (which optionally may include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme)) after 35 (CDR1), amino acid positions 50 to 65(CDR2), and amino acid positions 95 to 102(CDR 3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34(CDR1), amino acid positions 50 to 56(CDR2), and amino acid positions 89 to 97(CDR 3). In particular embodiments, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

In certain aspects, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of the structural loops of an immunoglobulin (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-. Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The ends of the Chothia CDR-HI loops when numbered using the Kabat numbering convention vary between H32 and H34 depending on the length of the loops (since the Kabat numbering scheme places the insertions at H35A and H35B; the loops end at 32 if neither 35A nor 35B are present; the loops end at 33 if only 35A is present; the loops end at 34 if both 35A and 35B are present). In particular embodiments, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

The terms "constant region" and "constant domain" are interchangeable and have the meaning common in the art. Constant regions are antibody portions, such as the carboxy-terminal portions of light and/or heavy chains, that are not directly involved in binding of the antibody to an antigen, but may exhibit various effector functions, such as interaction with an Fc receptor. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence relative to immunoglobulin variable domains.

When used in reference to an antibody, the term "heavy chain" constant domain-based amino acid sequence can refer to any of a variety of types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including the subclasses of IgG, e.g., IgG₁、IgG₂、IgG₃And IgG₄。

When used in reference to an antibody, the term "light chain" may refer to any of a variety of different types, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K) _D) And (4) showing. Affinity can be measured and/or expressed in a variety of ways known in the art, including but not limited to equilibrium dissociation constant (K)_D) And equilibrium association constant (K)_A)。K_DFrom k to k_off/k_onIs calculated by the quotient of K_AFrom k to k_on/k_offThe quotient of (2). k is a radical of_onRefers to, for example, the binding rate constant of an antibody to an antigen, and k_offRefers to, for example, dissociation of an antibody from an antigen. k is a radical of_onAnd k_offCan be prepared by techniques known to those of ordinary skill in the art such asOr a KinExA assay.

"conservative amino acid substitution" refers to an amino acid substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR or within a framework region of an antibody or antigen binding molecule thereof may be substituted with amino acid residues having similar side chains. In general, two sequences are generally considered "substantially similar" if they contain conservative amino acid substitutions at corresponding positions. For example, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or have "polar" or "nonpolar" side chains. Substitutions of one amino acid to another of the same type may be considered conservative substitutions. Exemplary amino acid classifications are summarized in tables 2 and 3 below:

TABLE 2

TABLE 3

Polysemous amino acid	3 letters	1 letter
			Asparagine or aspartic acid	Asx	B
Glutamine or glutamic acid	Glx	Z
			Leucine or isoleucine	Xle	J
Unspecified or unknown amino acids	Xaa	X

The term "heterologous" means from any source other than the naturally occurring sequence. For example, a heterologous sequence included as part of a co-stimulatory protein having the amino acid sequence of SEQ ID NO:232 (e.g., the corresponding human co-stimulatory protein) is an amino acid that does not naturally occur as (i.e., is not aligned with) the wild-type human co-stimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the wild-type human co-stimulatory protein coding sequence.

The term "epitope" is a term of art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can be, for example, two or more non-contiguous regions from a polypeptide or polypeptides (conformational, non-linear, non-contiguous, or non-contiguous epitopes). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, nuclear magnetic resonance spectroscopy (NMR spectroscopy), X-ray diffraction crystallographic studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Gieger R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University,1992, marketed by Molecular relations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114&115, eds Wyckoff HW et al; U.S.2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallog D Biol Crystallog 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roveri P et al, (2000) Acta Crystallog D Biol Crystallog 56(Pt 10 1321316) for example. Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M et al, (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

An antigen binding molecule, antibody or antigen binding molecule thereof "cross-competes" with a reference antibody or antigen binding molecule thereof if the interaction between the antigen and the first binding molecule, antibody or antigen binding molecule thereof blocks, limits, inhibits or otherwise reduces the ability of the reference binding molecule, reference antibody or antigen binding molecule thereof to interact with the antigen. The cross-competition may be complete, e.g. binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it may be partial, e.g. binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In certain embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds to the same or overlapping epitope as the reference antigen binding molecule. Many types of competitive binding assays can be used to determine whether one antigen binding molecule competes with another, for example: solid phase direct or indirect Radioimmunoassay (RIA); solid phase direct or indirect Enzyme Immunoassay (EIA); sandwich competition assays (Stahli et al, 1983, Methods in Enzymology 9: 242-; solid phase direct biotin-avidin EIA (Kirkland et al, 1986, J.Immunol.137: 3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (Harlow and Lane,1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA using a 1-125 labeled solid phase (Morel et al, 1988, mol. Immunol.25: 7-15); solid phase direct biotin-avidin EIA (Cheung, et al, 1990, Virology 176: 546-552); and direct markers RIA (Moldenhauer et al, 1990, Scand. J. Immunol.32: 77-82).

The term "binding" generally refers to a non-covalent association between two or more entities. Direct bonding involves physical contact between entities or moieties. "indirect" binding refers to physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of circumstances, for example, where interacting entities or moieties are studied in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and/or in a biological system (e.g., a cell)).

As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or an immune complex) as such binding is understood by those of skill in the art. For example, a molecule that specifically binds an antigen may bind to other peptides or polypeptides, usually with lower affinity, as by, for example, an immunoassay,Determined on a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In particular embodiments, a molecule that specifically binds an antigen is present at a higher K than when the molecule binds to another antigen _AAt least 2 log (log), 2.5 log, 3 log, 4 pairs greaterK of a number or more_ABinding to an antigen. Binding may comprise preferential association of a binding motif, antibody or antigen binding system with a target of the binding motif, antibody or antigen binding system as compared to association of the binding motif, antibody or antigen binding system with an entity that is not a target (i.e., a non-target). In some embodiments, a binding motif, antibody, or antigen binding system selectively binds to a target if the binding between the binding motif, antibody, or antigen binding system and the target is greater than 2-fold, greater than 5-fold, greater than 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or greater than 100-fold as compared to the binding of the binding motif, antibody, or antigen binding system and the non-target. In some embodiments, if the binding affinity is less than about 10^-5M, less than about 10^-6M, less than about 10^-7M, less than about 10^-8M, or less than about 10^-9M, then the binding motif, antibody or antigen binding system selectively binds to the target.

In another embodiment, the molecule that specifically binds to an antigen is at about 1x10^-7Dissociation constant (K) of M_d) And (4) combining. In some embodiments, when K _dIs about 1x10^-9M to about 5x10^-9M, the antigen binding molecule specifically binds to the antigen with "high affinity". In some embodiments, when K_dIs 1x10^-10M to about 5x10^-10M, the antigen binding molecule specifically binds to the antigen with "very high affinity". In one embodiment, the antigen binding molecule has a 10^-9K of M_d. In one embodiment, the off-rate is less than about 1x10^-5. In other embodiments, the antigen binding molecule is at about 1x10^-7M to about 1x10^-13K between M_dBinding to human BCMA. In yet another embodiment, the antigen binding molecule is at about l x10^-10M to about 5x10^-10K of M_dBinding to human BCMA. In some embodiments, the antigen binding molecule is at about 1x10^-7M and about 1x10^-13K between M_dBind to human CD19, CD20, or both. In yet another embodiment, the antigen binding molecule is at about 1x10^-10M toAbout 5x10^-10K of M_dBind to human CD19, CD20, or both.

In particular embodiments, provided herein are antibodies or antigen binding molecules thereof that bind to a target human antigen (e.g., human BCMA or human CLL-1) with higher affinity than another target antigen (e.g., non-human BCMA or non-human CLL-1). In some embodiments, provided herein are antibodies or antigen-binding molecules thereof that bind to human CD19, human CD20, or both, with higher affinity than another species of one or both target antigens, e.g., non-human CD19, non-human CD20, or both. In certain embodiments, provided herein are antibodies or antigen binding molecules thereof that bind to a target human antigen (e.g., human BCMA or human CLL-1) with an affinity that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher than another target antigen, as measured by, for example, a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In certain embodiments, provided herein are antibodies or antigen binding molecules thereof that bind to human CD19, human CD20, or both with an affinity 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher than another species of one or both target antigens, as measured by, for example, a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In particular embodiments, an antibody or antigen-binding molecule thereof described herein that binds to a target human antigen will bind to another target antigen with less than 10%, 15%, or 20% of the binding of the antibody or antigen-binding molecule thereof to the human antigen, as measured by, for example, a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

A "chimeric antigen receptor" or "CAR" refers to a molecule engineered to include a binding motif and a means to activate an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. In some embodiments, the CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR T cells. An "extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when present in a cell membrane, is understood to be located in the extracellular space outside of the cell membrane.

"antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production or activation of specific immunocompetent cells or both. One skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The antigen may be expressed endogenously, i.e. from genomic DNA, or may be expressed recombinantly. The antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly. In addition, larger molecule fragments may serve an antigenic role. In one embodiment, the antigen is a tumor antigen. In a specific embodiment, the antigen is all or a fragment of CD19 or CD 20. A "target" is any molecule bound by a binding motif, antigen binding system, or binding agent (e.g., an antibody). In some embodiments, the target is an antigen or epitope of the present disclosure.

The term "neutralizing" refers to an antigen binding molecule, scFv, antibody or fragment thereof that binds to a ligand and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof directly blocks a binding site on the ligand or alters the binding capacity of the ligand by an indirect means (e.g., a structural or energetic change in the ligand). In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof prevents the protein to which it binds from performing a biological function.

The term "autologous" refers to any substance derived from the same individual into which it is later reintroduced. For example, engineered autologous cell therapy (eACT) as described herein^TM) The method involves collecting lymphocytes from a patient, which are then engineered to express the examplesSuch as a CAR construct, and then administered back to the same patient.

The term "allogeneic" refers to any material that is derived from one individual and then introduced into another individual of the same species, such as allogeneic T cell transplantation.

The terms "transduction" and "transduced" refer to a process by which exogenous DNA is introduced into cells via a viral vector (see Jones et al, "Genetics: printers and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculovirus vector, an EB virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

"transformation" refers to any process of introducing foreign DNA into a host cell. Transformation can be performed using a variety of methods, either under natural or artificial conditions. Transformation can be accomplished using any known method for inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. In some embodiments, some transformation methods are selected based on the host cell being transformed and/or the nucleic acid to be inserted. Transformation methods may include, but are not limited to, viral infection, electroporation, and lipofection. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA is capable of replication as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cell can express the introduced nucleic acid.

The term "vector" refers to an acceptor nucleic acid molecule modified to contain or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors contain sequences that direct the expression of inserted genes, which are operably linked to these genes. Such vectors may be referred to herein as "expression vectors". Standard techniques can be used for engineering of vectors, for example, as found in Sambrook et al, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which are incorporated herein by reference for any purpose.

"cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or blood stream. "cancer" or "cancer tissue" may include tumors. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other leukocyte malignancies. In some embodiments, the methods of the present disclosure can be used to reduce tumor size, for example, from tumors of the following group: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, hodgkin's disease, non-hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), transformed follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), Chronic Lymphocytic Leukemia (CLL), solid tumors of childhood, lymphocytic lymphomas, bladder cancer, renal or ureteral cancer, renal pelvis cancer, Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), other B-cell malignancies, and combinations of said cancers. In a specific embodiment, the cancer is multiple myeloma. A particular cancer may be responsive to chemotherapy or radiation therapy or the cancer may be refractory. Refractory cancer refers to cancer that is not susceptible to surgical intervention, and that is either initially unresponsive to chemotherapy or radiation therapy, or that becomes unresponsive over time. Cancer further includes relapsed or refractory large B-cell lymphoma following two or more lineal therapies, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma following two or more lineal therapies, high grade B-cell lymphoma, and DLBCL caused by follicular lymphoma.

As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with the tumor. An anti-tumor effect may also refer to the prevention of tumorigenesis, e.g. a vaccine.

As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a particular antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. The cytokine may be expressed endogenously by a cell or administered to a subject. Cytokines can be released by immune cells (including macrophages, B cells, T cells, and mast cells) to spread the immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including Interleukins (IL)7 and IL-15, promote immune cell survival and proliferation, while pro-inflammatory cytokines can promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and Interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, Tumor Necrosis Factor (TNF) -alpha, TNF-beta, Fibroblast Growth Factor (FGF)2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1(sICAM-1), soluble vascular adhesion molecule 1(sVCAM-1), Vascular Endothelial Growth Factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme a, granzyme B, soluble Fas ligand (sFasL), and perforin (perforin). Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid a (saa).

"chemokines" are a class of cytokines that mediate cell chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1(MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10(IP-10), and thymus and activation regulated chemokine (TARC or CCL 17).

A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., an engineered CAR T cell) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by decreased severity of disease symptoms, increased frequency and duration of asymptomatic phases of the disease, or prevention of injury or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to skilled practitioners, for example in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by assaying the activity of the agent in an in vitro assay.

The term "lymphocyte" includes Natural Killer (NK) cells, T cells or B cells. NK cells represent the major component of the innate immune systemA cytotoxic (cell toxic) lymphocyte of the type (cytoxic). NK cells reject tumors as well as virus infected cells. It acts through the process of apoptosis or programmed cell death. It is called "natural killing" because it does not require activation to kill the cells. T cells play a major role in cell-mediated immunity (no participation of antibodies). Its T Cell Receptor (TCR) distinguishes itself from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem cell-like memory T cells_SCMThe cells (e.g., primary cells) are CD45RO-, CCR7+, CD45RA +, CD62L + (L-selectin), CD27+, CD28+, and IL-7 ra +, but they also express large amounts of CD95, IL-2R β, CXCR3, and LFA-1, and display unique functional attributes of many memory cells; (ii) central memory T _CM(ii) cells expressing L-selectin and CCR7 which secrete IL-2 but not IFN gamma or IL-4, and (iii) effector memory T_EMCells, however, do not express L-selectin or CCR7, but produce effector cytokines such as IFN γ and IL-4), regulatory T cells (Treg, suppressor T cells or CD4+ CD25+ regulatory T cells), natural killer T cells (NKT), and Gamma Delta T cells. On the other hand, B cells play a major role in humoral immunity (with antibody involvement). It generates antibodies and antigens and performs the action of Antigen Presenting Cells (APCs), and is transformed into memory B cells upon activation through antigen interaction. In mammals, immature B cells form in the bone marrow, which is the source of the B cell name.

By "linker" (L) or "linker domain" or "linker region" is meant an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which links together any domains/regions of the CARs of the invention. The linker may be composed of flexible residues such as glycine and serine so that adjacent protein domains may move freely with respect to each other. When it is desired to ensure that two adjacent domains do not lie spatiallyWhere they interfere, longer connectors may be used. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include a 2A linker (e.g., T2A), a 2A-like linker, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, a CHYSEL sequence of porcine teschovirus (P2A), a virus (T2A), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence may comprise Asp-Val/Ile-Glu-X-Asn-Pro-Gly. ^(2A)-Pro.^(2B)Motif (SEQ ID NO:314), which results in cleavage between 2A glycine and 2B proline. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention. For example, in some embodiments, a linker can be used to link or link two CARs of different antigen binding systems, such as a bicistronic CAR. The joint may be part of a multi-component agent connecting different components to each other. For example, a polypeptide comprising two or more functional domains or domains may comprise a stretch of amino acids between such domains that link them to each other. In some embodiments, the polypeptide comprising a linker element has the overall structure of general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains associated with each other through a linker. The linker may link or link together any domain/region of the CARs of the present disclosure. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, or 10 to 100 amino acids in length). In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

By "single chain variable fragment", "single chain antibody variable fragment" or "scFv" antibody is meant a form of antibody that comprises only heavy and light chain variable regions, which are linked by a linker peptide.

The term "genetically engineered" or "engineered" refers to methods of modifying the genome of a cell, including, but not limited to, deletions of coding or non-coding regions or portions thereof, or insertions of coding regions or portions thereof. In some embodiments, the modified cell is a lymphocyte (e.g., a T cell), which can be taken from a patient or donor. The cells can be modified to express exogenous constructs, such as Chimeric Antigen Receptors (CARs) or T Cell Receptors (TCRs) incorporated into the genome of the cells. Engineering typically involves manual operations. For example, a polynucleotide is considered "engineered" when two or more sequences that are not joined or linked together in that order in nature are manually manipulated to join or link directly to each other in the engineered polynucleotide. In the case of a cell manipulated by molecular biological techniques, a cell or organism is considered "engineered" (e.g., by introducing new genetic material that did not exist previously, e.g., by transformation, somatic hybridization, transfection, transduction, or other mechanism, or by altering or removing pre-existing genetic material, e.g., by substitution or deletion mutations, or by other protocols) if the cell or organism is manipulated such that its genetic information is altered. In some embodiments, the binding agent is a modified lymphocyte, e.g., a T cell, which can be obtained from a patient or donor. The engineered cells can be modified to express exogenous constructs, such as Chimeric Antigen Receptors (CARs) or T Cell Receptors (TCRs), that are integrated into the genome of the cells. Progeny of an engineered polynucleotide or binding agent are often referred to as "engineered", even if the actual operation is performed on a previous entity. In some embodiments, "engineered" refers to an entity that has been designed and produced. The term "designed" refers to the following agents: (i) the structure is manually selected or selected; (ii) it is generated by a process that requires manual labor; and/or (iii) it is different from natural substances and other known agents. "T cell receptor" or "TCR" refers to an antigen recognition molecule that is present on the surface of a T cell. During normal T cell development, each of the four TCR genes α, β, γ, and δ may rearrange, resulting in a highly diverse TCR protein.

By "immune response" is meant the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including abs, cytokines, and complements) that result in the selective targeting, binding, damaging, destroying, and/or excluding invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells in vertebrates, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or of recurrence of a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. The T cell therapy may include adoptive T cell therapy, Tumor Infiltrating Lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT)^TM) And allogeneic T cell transplantation. However, one skilled in the art will appreciate that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. patent publication nos. 2014/0154228 and 2002/0006409, U.S. patent No. 5,728,388, and international publication No. WO 2008/081035.

The T cells for immunotherapy may be from any source known in the art. For example, T cells may be differentiated from a population of hematopoietic stem cells in vitro, or T cells may be obtained from a subject. T cells can be obtained from, for example, Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, the T cells may be derived from one or more T cell lines available in the art. T cells can also be obtained using any number of techniques known to those skilled in the art (e.g., FICOLL)^TMSeparation and/or apheresis(apheresis)) was obtained from blood units collected from subjects. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.

The term "engineered autologous cell therapy" (which may be abbreviated as "eACT^TM", also known as adoptive cell transfer) is the process of collecting the patient's own T cells, which are then genetically altered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen, linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. The co-stimulatory domain may be derived from a naturally occurring co-stimulatory domain, e.g., having the amino acid sequence of SEQ ID NO:1 or a variant thereof (e.g., a variant having a truncated hinge domain ("THD")), while the activation domain may be derived from, e.g., CD 3-zeta. In certain embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. CAR scFv can be designed to target, for example, CD19, which CD19 is a transmembrane protein expressed by cells of the B cell lineage (including ALL normal B cell and B cell malignancies, including but not limited to NHL, CLL and non-T cell ALL). In some embodiments, the CAR is engineered such that the co-stimulatory domains are expressed as separate polypeptide chains. Example CAR T cell therapies and constructs are described in U.S. patent publication nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, which references are incorporated by reference herein in their entirety. "adoptive cell therapy" or "ACT" relates to the transfer of immune cells with anti-tumor activity to a subject, e.g., a cancer patient. In some embodiments, ACT is a therapeutic method involving the use of lymphocytes (e.g., engineered lymphocytes) having anti-tumor activity.

"patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). Herein, the terms "subject" and "patient" are used interchangeably.

The term "in vitro" refers to events occurring in an artificial environment, such as in a test tube, reaction vessel, cell culture, etc., rather than in a multicellular organism. The term "in vitro cell" refers to any cell cultured in vitro. In particular, the in vitro cells may comprise T cells. The term "in vivo" refers to an event that occurs within a multicellular organism, such as a human or non-human animal.

An "antigen specific targeting region" (ASTR) refers to a region of a CAR that targets a particular antigen. The CAR of the invention comprises at least two targeting regions that target at least two different antigens. In one embodiment, the CAR comprises three or more targeting regions that target at least three or more different antigens. The targeted region on the CAR is extracellular. In some embodiments, the antigen specific targeting region comprises an antibody or functional equivalent thereof or fragment or derivative thereof, and each targeting region targets a different antigen. The targeting region can comprise a full-length heavy chain, a Fab fragment, a single-chain fv (scfv) fragment, a bivalent single-chain antibody, or a diabody, each of which is specific for a target antigen. However, there are many alternatives to promote an immune response, such as linked cytokines (which result in the recognition of cells bearing cytokine receptors), affibodies, ligand binding domains from naturally occurring receptors, soluble protein/peptide ligands of receptors (e.g., on tumor cells), peptides and vaccines, each of which can be used in various embodiments of the invention. In fact, as will be appreciated by those skilled in the art, almost any molecule that binds a given antigen with high affinity can be used as an antigen-specific targeting region.

"antigen presenting cell" or "APC" refers to a cell that processes and presents an antigen to a T cell. Exemplary APCs include dendritic cells, macrophages, B cells, certain activated epithelial cells, and other cell types capable of TCR stimulation and appropriate T cell co-stimulation.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. There is no limit to the maximum number of amino acids that a protein or peptide contains at least two amino acids and may comprise the sequence of the protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which are of many types). "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its associated ligand, wherein the binding mediates a signaling event. "stimulating molecule" refers to a molecule on a T cell, such as the T Cell Receptor (TCR)/CD3 complex that specifically binds to a cognate stimulating ligand present on an antigen presenting cell. "stimulatory ligand" refers to a ligand that, when present on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, etc.), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response (including but not limited to activation, initiation of an immune response, proliferation, etc.) of the T cell. Stimulatory ligands include, but are not limited to, anti-CD 3 antibodies (e.g., OKT3), MHC class I molecules loaded with peptides, hyperactivating anti-CD 2 antibodies, and hyperactivating anti-CD 28 antibodies.

As used herein, "co-stimulatory signal" refers to a signal that, when combined with a primary signal (e.g., TCR/CD3 linkage), results in a T cell response (e.g., without limitation, up-or down-regulation of proliferation and/or key molecules).

As used herein, "co-stimulatory ligand" includes molecules on antigen presenting cells that specifically bind to cognate co-stimulatory molecules on T cells. Binding of the co-stimulatory ligand provides a signal that mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc. The co-stimulatory ligand induces a signal in addition to the primary signal provided by the stimulatory molecule, e.g., provided by the binding of the T Cell Receptor (TCR)/CD3 complex to a Major Histocompatibility Complex (MHC) molecule loaded with a peptide. Costimulatory ligands can include, but are not limited to, 3/TR6, 4-1BB ligand, agonists or antibodies that bind Toll ligand receptors, B7-1(CD80), B7-2(CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus invasion medium (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), ligands that specifically bind B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or Programmed Death (PD) L1. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligands that specifically bind to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14(TNFSF14 or LIGHT).

A "costimulatory molecule" is an associated binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., without limitation, proliferation) of the T cell. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD2, CD276 (B2-H2), CD2 (alpha; delta; epsilon; gamma; zeta), CD2, CD49 2, CD 368, CD 6372, CD 72, CD2, GAI-2, CD-C-I-L2, CD-L-2, CD-L2, CD-L (GAI-L (CD-L-2, CD-L-2, CD-L (CD-2, CD-L-2, CD-L (CD-L-2, CD-L (CD-L-2, CD-L (CD-L-2, CD-L (CD-L, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecule, SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4), SLAMF6 (VLB-24; VLSLLA), SLNL 5976, TNFR 596, TNFR 599, TNFR 596, or a truncated fragment of a receptor thereof.

Herein, the terms "reduce" and "reducing" are used interchangeably and mean any change less than the original. "reduction" and "decrease" are relative terms and require comparison between before and after measurement. "reduce" and "reducing" include complete depletion.

The terms "improve," "increase," "inhibit," and "decrease" refer to a value measured relative to a baseline or other reference. In some embodiments, an appropriate reference measurement may comprise a measurement in an otherwise equivalent condition in the absence of an agent or treatment (e.g., before and/or after), or in certain systems (e.g., a single individual) in the presence of an appropriate equivalent reference agent. In some embodiments, an appropriate reference measurement may comprise a measurement in an equivalent system that is known or expected to respond in an equivalent manner in the presence of the relevant agent or treatment.

By "treating" or "treatment" of a subject is meant any type of intervention or process performed on the subject, or administering an active agent to the subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, severity, or recurrence of a symptom, complication, or condition, or biochemical indicator associated with the disease. In one embodiment, "treating" or "treatment" includes partial remission. In another embodiment, "treating" or "treatment" includes complete remission. In some embodiments, treatment may be for subjects who do not exhibit signs of the relevant disease, disorder, and/or condition and/or subjects who exhibit only early signs of the disease, disorder, and/or condition. In some embodiments, such treatment may be directed to a subject exhibiting one or more defined signs of the associated disease, disorder, and/or condition. In some embodiments, the treatment may be directed to a subject who has been diagnosed with the relevant disease, disorder, and/or condition. In some embodiments, treatment may be directed to a subject known to have one or more susceptibility factors statistically associated with the relevant disease, disorder, and/or condition.

The term "agent" may refer to any class of molecules or entities comprising, or to a plurality of molecules or entities, any of which may serve as: such as polypeptides, nucleic acids, carbohydrates, lipids, small molecules, metals, cells or organisms (e.g., fractions or extracts thereof), or components thereof. In some embodiments, the agent may be used in isolated or pure form. In some embodiments, the agent may be used in crude or impure form. In some embodiments, the agents may be provided as a population, collection, or library, which may be screened, for example, to identify or characterize the members present therein.

Two events or entities are "associated" with each other if the presence, level, and/or form of one event or entity is associated with the other. For example, an entity (e.g., a polypeptide, genetic signature (signature), metabolite, microorganism, etc.) is considered associated with a disease, disorder, or condition if its presence, level, and/or form is associated with the occurrence and/or susceptibility to the disease, disorder, or condition (e.g., in a relevant population). For example, two or more entities are "associated" with each other physically if they interact, directly or indirectly, such that they are in physical proximity to each other and/or remain in physical proximity (e.g., bind). In further examples, two or more entities that are physically associated with each other are covalently linked or linked to each other, or non-covalently associated, such as through hydrogen bonding, van der waals interactions, hydrophobic interactions, attractive forces, and combinations thereof.

The term "identity" refers to the overall relatedness between polymeric molecules, for example between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Methods for calculating percent identity between two provided polypeptide sequences are known. Calculation of percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at the corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps and the length of each gap (which may need to be introduced to achieve optimal alignment of the two sequences). Comparison or alignment of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as BLAST (basic local alignment search tool). In some embodiments, polymer molecules are considered "homologous" to each other if the sequence of the polymer molecules is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

To calculate percent identity, the sequences being compared are typically aligned in a manner that results in the greatest match between the sequences. One example of a Computer program that can be used to determine percent identity is the GCG package, which includes GAP (Devereux et al, 1984, Nucl. acid Res.12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for percent sequence identity to be determined. The sequences are aligned so that their respective amino acids or nucleotides match best (e.g., "matched spans" as determined by an algorithm). In certain embodiments, the algorithm also uses a standard comparison matrix (see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure 5: 345-. Other algorithms may also be used to compare amino acid or nucleic acid sequences, including algorithms available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such procedures are described in Altschul, et al, Basic local alignment search tool, J.mol.biol.,215(3): 403-; altschul, et al, Methods in Enzymology; altschul, et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res.25:3389-3402, 1997; baxevanis, et al, Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press, 1999. In addition to identifying similar sequences, the above procedures generally provide an indication of the degree of similarity. In some embodiments, two sequences are considered substantially similar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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% or more of the corresponding residues of the two sequences are similar and/or identical in the corresponding stretch of residues (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the respective segment is a complete sequence. In some embodiments, the respective segment is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, or more residues. Sequences having substantial sequence similarity may be homologs of each other.

"combination therapy" refers to a condition in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more treatment moieties). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administering" of a combination therapy may involve administering one or more agents or modalities to a subject that is receiving the other agents or modalities in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments two or more agents, or active portions thereof, may be used together in a combined composition, or even in a combined compound (e.g., as part of a single chemical complex or covalent entity).

"corresponding to" can be used to specify the position/identity of a structural element in a molecule or composition by comparison with an appropriate reference molecule or composition. For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be identified as "corresponding" to a residue in an appropriate reference polymer. For example, for simplicity, a canonical numbering system based on a reference related polypeptide may be used to designate a residue in the polypeptide such that the amino acid "corresponding to" the residue at position 100, for example, need not actually be the 100 th amino acid in the amino acid chain, so long as it corresponds to the residue seen at position 100 in the reference polypeptide. Various sequence alignment strategies are available, including software programs such as, for example, BLAST, CS-BLAST, CUDASW + +, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Inferal, KLAST, USEARCH, paramail, PSI-BLAST, PSI-Search, ScaLABLAST, Sequila, SAM, SSEARCH, SWAPHI-LS, SWIMM, or SWIPE, which can be used, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids according to the present disclosure.

The term "domain" refers to a portion of an entity. In some embodiments, a "domain" is associated with a structural and/or functional feature of an entity, e.g., such that when the domain is physically separated from the rest of its parent entity, it substantially or completely retains the structural and/or functional feature. In some embodiments, a domain may comprise a portion of an entity that, when separated from the (parent) entity and connected or linked to a different (recipient) entity, substantially retains and/or confers one or more structural and/or functional characteristics to the recipient entity, e.g., its characteristics in the parent entity. In some embodiments, a domain is part of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, the domain is a portion of a polypeptide; in some such embodiments, a domain is characterized by a structural element (e.g., an amino acid sequence or sequence motif, an alpha-helical feature, a beta-sheet feature, a coiled-coil feature, a random coil feature, etc.), and/or by a functional feature (e.g., a binding activity, an enzymatic activity, a folding activity, a signaling activity, etc.).

The term "dosage form" may be used to refer to a physically discrete unit of an active agent (e.g., an antigen binding system or an antibody) for administration to a subject. Typically, each such unit contains a predetermined amount of active agent. In some embodiments, such amounts are those amounts (or all fractions thereof) of a unit dose suitable for administration according to a dosing regimen that, when administered to a relevant population, has been determined to be associated with a desired or beneficial result. The total amount of therapeutic composition or agent administered to a subject is determined by one or more medical practitioners and may involve the administration of more than one dosage form.

The term "dosing regimen" can be used to refer to a set of one or more unit doses administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen comprises a plurality of doses, wherein each dose is separated in time from the other doses. In some embodiments, the dosing regimen comprises a plurality of doses and consecutive doses are spaced from each other by a period of equal length; in some embodiments, the dosing regimen comprises multiple doses and consecutive doses are separated from each other by at least two time periods of different lengths. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen comprises a first dose having an amount of a first dose, followed by one or more additional doses having an amount of a second dose that is different from the amount of the first dose. In some embodiments, the dosing regimen is adjusted periodically to achieve a desired or beneficial result.

"Effector function" refers to the biological consequences of an antibody Fc region interacting with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). The effector function may be antigen binding dependent, antigen binding independent, or both. ADCC refers to the lysis of antibody-binding target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve effector cells carrying Fc receptors (fcrs) recognizing and subsequently killing antibody-coated target cells (e.g., cells expressing antibody-bound antigen on their surface). Effector cells that mediate ADCC may comprise immune cells, including but not limited to one or more of Natural Killer (NK) cells, macrophages, neutrophils, eosinophils.

"Effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, the effector cells may include, but are not limited to, one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, langerhans cells, Natural Killer (NK) cells, T lymphocytes, and B lymphocytes. The effector cells may be of any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.

The term "excipient" refers to an agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

A "fragment" or "portion" of a material or entity as described herein has a structure that comprises a discrete portion of the whole, e.g., a discrete portion of a physical entity or an abstract entity. In some embodiments, a fragment lacks one or more portions found in the entirety. In some embodiments, a fragment consists of or comprises a characteristic structural element, domain or portion found in the whole. In some embodiments, the polymer segment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the overall polymer. In some embodiments, a polymer segment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of monomeric units (e.g., residues) (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) as found in the overall polymer. In some embodiments, the monolithic material or entity may be referred to as the "parent" of the fragment.

The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. In general, a polypeptide containing at least two such segments is considered a fusion polypeptide if the two segments are (1) portions that are not naturally contained in the same peptide, and/or (2) portions that were not previously linked or linked in a single polypeptide, and/or (3) portions that are linked or linked to each other by artificial action.

The term "gene product" or "expression product" generally refers to an RNA transcribed from a gene (before and/or after processing) or a polypeptide encoded by an RNA transcribed from a gene (before and/or after modification).

The term "isolated" refers to a substance that (1) has been separated from at least some components with which it was associated at an earlier time or with which it would otherwise be associated, and/or (2) is present in a composition that contains a limited or defined amount or concentration of one or more known or unknown contaminants. In some embodiments, an isolated species may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of other non-species components (which are associated with the species at an earlier time, e.g., other components or contaminants that the species was or would otherwise be associated with). In certain instances, a substance is isolated if it is present in a composition that contains a limited or reduced amount or concentration of the same or similar type of molecule. For example, in certain instances, a nucleic acid, DNA, or RNA species is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of the non-species nucleic acid, DNA, or RNA molecule. For example, in certain instances, a polypeptide species is isolated if it is present in a composition that contains a limited or reduced amount or concentration of polypeptide molecules that are not the species. In certain embodiments, the amount can be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, a limited amount can be an amount that does not exceed 100% of the amount of a substance in the composition, such as not exceeding 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of the amount of a substance in the composition. In some cases, the composition is pure or substantially pure for the selected substance. In some embodiments, the isolated material is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). A substance is "pure" if it is substantially free of other components or free of contaminants. In some embodiments, a substance may still be considered "isolated" or even "pure" after combination with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the material is calculated without such carriers or excipients.

"nucleic acid" refers to any polymeric strand of nucleotides. The nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid comprises one or more native nucleic acid residues. In some embodiments, the nucleic acid comprises one or more nucleic acid analogs. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) based on polymerization of complementary templates, propagation in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, the nucleic acid is partially or fully single stranded; in some embodiments, the nucleic acid is partially or fully double stranded. In some embodiments, the nucleic acid has a nucleotide sequence that comprises at least one element encoding a polypeptide or is the complement of a sequence encoding a polypeptide.

"operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a control element that is "operably linked" to a functional element is associated with a means for effecting the expression and/or activity of the functional element under conditions compatible with the control element.

The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or provides a benefit to the recipient thereof over any deleterious effect. With respect to the carrier, diluent or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or the benefit to the recipient thereof outweighs any deleterious effect. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting an agent from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient, or the benefit to the recipient thereof must outweigh any deleterious effects. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible materials for use in pharmaceutical formulations.

The term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to an associated subject or population, displays a statistically significant probability of achieving the intended therapeutic effect. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, including but not limited to forms suitable for: oral administration, such as drench (aqueous or non-aqueous solution or suspension), tablets, such as those for buccal, sublingual and systemic absorption, pills, powders, granules, pastes for application to the tongue; parenteral administration, for example by subcutaneous, intramuscular, intravenous or epidural injection, as for example sterile solutions or suspensions, or sustained release formulations; topical application, e.g. as a cream, ointment or controlled release patch or spray, to the skin, lung or oral cavity; intravaginally or intrarectally, e.g., as pessaries, creams or foams; under the tongue; an eye portion; transdermal; or nose, lung, and other mucosal surfaces.

The term "reference" describes a standard or control to which it is compared. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, a reference or control is tested, measured, and/or assayed substantially simultaneously with a test, measurement, or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, the reference or control is determined or characterized under conditions or circumstances comparable to those of the subject under evaluation. When there is sufficient similarity to justify dependence and/or comparison to a selected reference or control.

"regulatory T cells" ("Tregs", "Treg cells" or "Tregs") refer to the CD4+ T lymphocyte lineage involved in controlling certain immune activities such as autoimmunity, allergy and response to infection. Regulatory T cells can regulate the activity of T cell populations and may also affect certain innate immune system cell types. Tregs can be identified by expression of the biomarkers CD4, CD25 and Foxp3 and low expression of CD 127. Naturally occurring Treg cells typically account for 5-10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor infiltrating Treg cells) may account for up to 20-30% of the total population of CD4+ T lymphocytes.

The term "sample" generally refers to an aliquot of a material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may comprise a cell or organism, such as a population of cells, a tissue, or an animal (e.g., a human). In some embodiments, the source of interest comprises a biological tissue or fluid. In some embodiments, the biological tissue or fluid may comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculatory fluid, endolymph, exudate, stool, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum, semen, serum, periderm scale, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and/or combinations or components thereof. In some embodiments, the biological fluid may comprise intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and/or transcellular fluid. In some embodiments, the biological fluid may comprise plant exudate. In some embodiments, the biological tissue or sample can be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing, or lavage (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other irrigation or lavage). In some embodiments, the biological sample comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, it will be clear from the context that the term "sample" refers to a preparation obtained by processing (e.g., by removing one or more components and/or by adding one or more pharmaceutical agents thereto) a primary sample. Such "processed samples" may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acids, isolation and/or purification of certain components, and the like.

The term "cancer stage" refers to a qualitative or quantitative assessment of the level of cancer progression. In some embodiments, criteria for determining the stage of a cancer may include, but are not limited to, one or more of the location of the cancer in the body, the size of the tumor, whether the cancer has spread to lymph nodes, whether the cancer has spread to one or more different sites in the body, and the like. In some embodiments, the cancer may be staged using the so-called TNM system, according to which T refers to the size and extent of the primary tumor (often referred to as the primary tumor); n refers to the number of nearby lymph nodes with cancer; and M refers to whether the cancer has metastasized. In some embodiments, the cancer may be referred to as stage 0 (abnormal cells are present but not spread to nearby tissues, also known as carcinoma in situ or CIS; CIS is not cancer but may become cancerous), stage I-III (cancer is present; the larger the number, the larger the tumor, the higher the extent of spread to nearby tissues), or stage IV (cancer spreads to distant sites in the body). In some embodiments, the cancer may be assigned to a stage selected from the group consisting of: in situ; localization (cancer is limited to where it begins, with no signs of spread); regional (cancer has spread to nearby lymph nodes, tissues or organs); distant (cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the phase).

The phrase "therapeutic agent" may refer to any agent that causes a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect in a suitable population. In some embodiments, a suitable population may be a population of model organisms or human subjects. In some embodiments, a suitable population may be defined by various criteria, such as a particular age group, gender, genetic background, pre-existing clinical condition, presence or absence according to biomarkers, and the like. In some embodiments, a therapeutic agent is a substance that can be used to reduce, ameliorate, alleviate, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, the therapeutic agent is an agent that has been or needs to be approved by a governmental agency before it can be marketed for administration to a human. In some embodiments, the therapeutic agent is a medicament that requires a medical prescription to be administered to a human.

Various aspects of the disclosure are described in further detail in the following subsections. The present disclosure provides antigen binding systems and binding agents comprising at least an anti-CD 20 binding motif. Among other things, the present disclosure provides methods and compositions for treating cancer and/or for initiating or modulating an immune response. In certain embodiments, the disclosure encompasses dual targeting antigen binding systems and binding agents in that they comprise an anti-CD 20 binding motif and a second binding motif for a second antigen or epitope. In some cases, the second binding motif selectively binds CD 19. In various embodiments, one or more binding motifs is an scFv. Provided herein are exemplary binding motif amino acid sequences and nucleic acid sequences encoding the same. In some embodiments, the antigen binding system of the present disclosure is a chimeric antigen receptor. In some embodiments, the antigen binding systems of the present disclosure are bispecific or bicistronic chimeric antigen receptors. In some embodiments, the binding agents of the present disclosure are engineered T cell receptors.

Various embodiments of the present disclosure provide vectors encoding the binding motifs or antigen binding systems provided herein, for example vectors encoding anti-CD 20/anti-CD 19 antigen binding systems, such as bispecific or bicistronic anti-CD 20/anti-CD 19 chimeric antigen receptors. Various embodiments of the present disclosure provide binding agents that are cells that encode or express the antigen binding systems or binding motifs provided herein, e.g., T cells engineered to encode or express an anti-CD 20/anti-CD 19 chimeric antigen receptor, such as a bispecific or bicistronic anti-CD 20/anti-CD 19 chimeric antigen receptor. The present disclosure provides binding agents, e.g., binding agents comprising an immune cell that is genetically modified with an integrated gene, e.g., a nucleotide sequence of interest (e.g., a constitutive expression construct and/or an inducible expression construct comprising such a nucleotide sequence). In some embodiments, the present disclosure provides a method of treating a subject having a tumor comprising administering to the subject a binding agent therapy described herein and/or a protein therapeutic described herein. In some embodiments, the methods further comprise administering one or more additional therapies (e.g., a second binding agent described herein (e.g., CAR-T cells, CAR-NK cells, TCR-T cells, TIL cells, allogeneic NK cells, and autologous NK cells)), an antibody-drug conjugate, an antibody, a bispecific antibody, a T cell-engaging bispecific antibody, an engineered antibody, and/or a polypeptide).

Other features, objects, and advantages of the disclosure will be apparent from the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, not limitation.

The anti-CD 20 binding motifs of the present disclosure may comprise antigen binding sequences as found in the antibodies described herein. In some cases, an anti-CD 20 binding motif of the present disclosure comprises an antigen binding fragment described herein. Unless otherwise indicated, reference to CD20 in this disclosure is understood to refer to human CD 20. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise at least one heavy chain cdr (HCDR) provided herein, e.g., at least one HCDR disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise at least one light chain cdr (LCDR) provided herein, e.g., at least one LCDR disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise two LCDRs provided herein, e.g., at least two LCDRs disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises three LCDRs provided herein, e.g., three LCDRs disclosed in any one of tables 4-13.

In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise at least one HCDR provided herein, e.g., as disclosed in any one of tables 4-13, and at least one LCDR provided herein, e.g., as disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise one HCDR provided herein, e.g., at least one HCDR disclosed in any one of tables 4-13, and one LCDR provided herein, e.g., derived from the same table in tables 4-13 as the HCDR. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in any of tables 4-13, and two LCDRs provided herein, e.g., at least two LCDRs disclosed in any of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in any one of tables 4-13, and two LCDRs provided herein, e.g., derived from the same table in tables 4-13 as the HCDRs. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in any one of tables 4-13, and three LCDRs provided herein, e.g., three LCDRs disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in any one of tables 4-13, and three LCDRs derived from the same table in tables 4-13 as the HCDRs.

In various embodiments, the anti-CD 20 binding motifs of the present disclosure comprise at least one heavy chain framework region (heavy chain FR) of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR chain of a heavy chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in any one of tables 4-13.

In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of tables 4-13.

In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises at least one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in any one of tables 4-13, and at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in any one of tables 4-13, and one light chain FR of a light chain variable domain disclosed herein, e.g., derived from the same table in tables 4-13 as the heavy chain FR. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of tables 4-13, and two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of tables 4-13, and two light chain FRs of a light chain variable domain disclosed herein, e.g., derived from the same tables in tables 4-13 as the heavy chain FRs. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in any one of tables 4-13, and three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, the anti-CD 20 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of tables 4-13, and three light chain FRs derived from the same tables in tables 4-13 as the heavy chain FRs.

The exemplary antibody sequences provided in tables 4-13 are applicable to any antibody format, including, for example, a tetrameric antibody, a monospecific antibody, a bispecific antibody, an antigen binding fragment, or a binding motif. The heavy and light chain variable domains provided in tables 4-13, and portions thereof, can be included in the binding motif.

In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises one, two, or three FRs that together or each individually are at least 75% identical (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the FRs of a corresponding heavy chain variable domain of a heavy chain variable domain disclosed in any one of tables 4-13. In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises one, two, or three FRs that together or each individually are at least 75% identical (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%) to the FRs of a corresponding light chain variable domain of a light chain variable domain disclosed in any one of tables 4-13.

In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of tables 4-13. In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises two heavy chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of tables 4-13, which heavy chain variable domains may be the same or different.

In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises two light chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of tables 4-13, which light chain variable domains may be the same or different.

In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of tables 4-13, and at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of tables 4-13. In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises a heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of tables 4-13, and a light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of tables 4-13, optionally derived from the same as in tables 4-13 Table (7).

In various embodiments, an anti-CD 20 binding motif of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of tables 4-13, and two light chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of tables 4-13, wherein in various embodiments, (i) each heavy chain variable domain may be the same or different; (ii) each light chain variable domain may be the same or different; (iii) the at least one heavy chain variable domain and the at least one light chain variable domain may be derived from the same table in tables 4-13; or (iv) both heavy chain variable domains and both light chain variable domains are derived from the same table in tables 4-13. Each of tables 4-13 represents the heavy chain variable domain and light chain variable domain sequences of an exemplary antibody comprising (i) the heavy chain variable domain of an exemplary antibody; (ii) a DNA sequence encoding a heavy chain variable domain; (iii) three heavy chain variable domain CDRs of the heavy chain variable domain numbered according to IMGT, Kabat and Chothia; (iv) a light chain variable domain of an exemplary antibody; (v) a DNA sequence encoding a light chain variable domain; and (vi) three light chain variable domain CDRs of the light chain variable domain numbered according to IMGT, Kabat and Chothia. The information provided in each table provides the framework amino acid sequences, as well as the nucleotide sequences encoding each CDR amino acid sequence and the nucleotide sequences encoding the corresponding FR amino acid sequences.

In various embodiments, a binding motif can comprise a heavy chain variable domain of the disclosure (e.g., which has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain in any of tables 4-13, a light chain variable domain of the disclosure (e.g., which has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% to a light chain variable domain in any of tables 4-13) and a linker (e.g., a linker according to SEQ ID NO:247 and/or a linker according to any of SEQ ID NO: 307-313) A joint; see, e.g., Whitlow et al protein Eng.1993 Nov; 6(8):989-95). In various embodiments, the binding motif can comprise a leader sequence, a heavy chain variable domain of the present disclosure (e.g., which has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain in any of tables 4-13, a light chain variable domain of the present disclosure (e.g., which has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identity, to a light chain variable domain in any of tables 4-13), and a linker. If an amino acid or nucleotide sequence is provided that comprises a heavy chain variable domain of the present disclosure and a binding motif for a light chain variable domain of the present disclosure, a linker joining the two variable domains will be apparent from the sequence based on the present disclosure. A leader sequence will be apparent from the present disclosure if an amino acid or nucleotide sequence is provided that comprises a heavy chain variable domain of the present disclosure and a binding motif for a light chain variable domain of the present disclosure. For the avoidance of doubt, the heavy and light chain variable domains of the invention may be present in any orientation, for example an orientation in which the heavy chain variable domain is C-terminal to the light chain variable domain or in which the heavy chain variable domain is N-terminal to the light chain variable domain. In various embodiments, the binding motif may comprise a linker according to SEQ ID NO 247 and/or a linker according to any one of SEQ ID NO 307-313 adjacent to one or more further linkers.

In certain embodiments, an anti-CD 20 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 247. In certain embodiments, the anti-CD 20 binding motifs of the present disclosure comprise a binding motif comprising a linker according to SEQ ID NO:247 and/or a linker according to any one of SEQ ID NO: 307-313. In certain embodiments, an anti-CD 20 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 245. In certain embodiments, an anti-CD 20 binding motif of the present disclosure comprises a binding motif comprising the CSF2RA leader sequence according to SEQ ID NO: 245. In certain embodiments, the anti-CD 20 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, a linker of the present disclosure, and a leader sequence of the present disclosure. Exemplary nucleotide sequences encoding the anti-CD 19 binding motif and components thereof are found in SEQ ID NOs 246 and 248. In various embodiments, the binding motifs of the present disclosure have a sequence according to any one of the sequences of Table 53 (SEQ ID NO: 251-260).

Binding agents of the present disclosure based on the exemplary antibodies provided herein (such as, e.g., Ab1) can be provided in any fragment or form comprising a heavy chain variable domain according to the illustrated exemplary antibody and a light chain variable domain according to the illustrated exemplary antibody.

Table 4: exemplary antibody sequence 1(Ab1)

Table 5: exemplary antibody sequence 2(Ab2)

Table 6: exemplary antibody sequence 3(Ab3)

Table 7: exemplary antibody sequence 4(Ab4)

Table 8: exemplary antibody sequence 5(Ab5)

Table 9: exemplary antibody sequence 6(Ab6)

Table 10: exemplary antibody sequence 7(Ab7)

Table 11: exemplary antibody sequence 8(Ab8)

Table 12: exemplary antibody sequence 9(Ab9)

Table 13: exemplary antibody sequence 10(Ab10)

The present disclosure encompasses antibodies and antigen binding systems comprising an anti-CD 20 binding motif and a second binding motif that binds to a second target antigen or epitope, e.g., an antigen that is not CD20 (e.g., CD 19). The dual-targeted antigen-binding system comprises a bispecific CAR and a bicistronic CAR. Many antigen binding motifs are known. In various embodiments, the second target antigen is CD 19. The present specification encompasses a variety of second target antigens including, but not limited to, second target antigens that are 5T4, alpha-fetoprotein, B Cell Maturation Antigen (BCMA), CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD56, CD123, CD138, C-Met, CSPG4, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, 46gd 48, bound HER1-HER2, bound HER2-HER3, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp 9, HIV-1 envelope glycoprotein gpl20, IL-llrall, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, VEGFR 8653, prostate specific antigen or a combination thereof. Accordingly, in various embodiments, an antigen binding system or antibody of the present disclosure may comprise a first binding motif that is an anti-CD 20 binding motif and a second binding motif that is a second binding motif of 5T4, alpha fetoprotein, B Cell Maturation Antigen (BCMA), CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD56, CD123, CD138, C-Met, CSPG4, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, bound HER 42-HER 2, bound HER2-HER3, HER 56/Neu, HERV-K, HIV-1 envelope glycoprotein gp 27, HIV-1 envelope glycoprotein gpl20, IL-llRalpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-82867, VEGFR 7, VEGFR2, or a specific combination thereof. In some embodiments, the second antigen is an antigen characteristic of a B cell or a subset thereof, optionally wherein the second antigen is not CD19 or CD 20. Examples of binding motifs targeting these second antigens are known and/or provided herein.

In some cases, in an antigen binding system, such as a bispecific CAR, comprising an anti-CD 20 binding motif (e.g., comprising a heavy chain variable domain and/or a light chain variable domain of the present disclosure) and an anti-CD 19 binding motif (e.g., comprising a heavy chain variable domain and/or a light chain variable domain of the present disclosure), the anti-CD 20 binding motif (or a heavy chain variable domain and/or a light chain variable domain thereof) is closer to the C-terminus of the chimeric antigen receptor than the anti-CD 19 binding motif (or a heavy chain variable domain and/or a light chain variable domain thereof). In some cases, in an antigen binding system, such as a bispecific CAR, comprising an anti-CD 20 binding motif (e.g., comprising a heavy chain variable domain and/or a light chain variable domain of the present disclosure) and an anti-CD 19 binding motif (e.g., comprising a heavy chain variable domain and/or a light chain variable domain of the present disclosure), the anti-CD 20 binding motif (or a heavy chain variable domain and/or a light chain variable domain thereof) is closer to the N-terminus of the agent than the anti-CD 19 binding motif (or a heavy chain variable domain and/or a light chain variable domain thereof).

CD19 (also known as cluster of differentiation 19, B lymphocyte antigen CD19, B lymphocyte surface antigen B4, B4, CVID3, differentiation antigen CD19) is a protein encoded by the CD19 gene in humans. Unless otherwise indicated, reference to CD19 in this disclosure is understood to refer to human CD 19. It is found on the surface of B cells. Since CD19 expresses a B cell marker, it can be used as an antigen, for example, to recognize B cells and B cell derived cancer cells, such as B cell lymphoma. anti-CD 19 antibodies can bind to CD19 expressed in, for example, B lymphocytes in peripheral blood and spleen, B-cell chronic lymphocytic leukemia (B-CLL) cells, prolymphocytic leukemia (PLL) cells, Hairy Cell Leukemia (HCL) cells, Common Acute Lymphoblastic Leukemia (CALL) cells, pre-B acute lymphoblastic leukemia (pre-B-ALL) cells, and NULL acute lymphoblastic leukemia (NULL-ALL) cells to provide some non-limiting examples. An exemplary pharmaceutical product comprising an antigen binding system comprising an anti-CD 19 binding motif is a pharmaceutical product Is a CD 19-directed genetically modified autologous T cell immunotherapy useful for treating adult patients with relapsed or refractory large B cell lymphoma after two or more lines of systemic therapy, including Diffuse Large B Cell Lymphoma (DLBCL), primary mediastinal large B cell lymphoma, high grade B cell lymphoma, and DLBCL caused by follicular lymphoma (see also, e.g., CD 19-directed genetically modified autologous T cell immunotherapy for treating adult patients with relapsed or refractory large B cell lymphoma after two or more lines of systemic therapyFDA approved package insert, methods and compositions relating to immunotherapy, the entire contents of which are incorporated herein by reference). Comprising anti-CD 19 bindingAnother exemplary pharmaceutical product of the antigen binding system of motifs is a pharmaceutical productIs a CD 19-directed genetically modified autologous T cell immunotherapy useful for the treatment of: (1) patients under 25 years of age with refractory or second or later relapsed B-cell precursor Acute Lymphoblastic Leukemia (ALL); and (2) adult patients with relapsed or refractory (r/r) large B-cell lymphoma following two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma, and DLBCL caused by follicular lymphoma, not otherwise specified (see figure for reference)FDA approved package insert, incorporated herein by reference in its entirety for methods and compositions related to immunotherapy).

Andboth comprise an antibody binding domain derived from an anti-human CD19 antibody. Many anti-CD 19 antibodies are thought to bind to the epitope of CD19 encoded in exon 4 of the CD19 gene. Other anti-CD 19 binding motifs may recognize different epitopes of CD19, or the same epitope with different affinities. The antigen binding system may comprise an antigen binding domain derived from, for example, SJ25C 1. The CD19 antibody, clone SJ25C1, was derived from the hybridization of Sp2/0 mouse myeloma cells with spleen cells isolated from BALB/C mice immunized with NALM1 and NALM16 cells. SJ25C1 antigen binding domains were used for other investigational CD19 targeted Chimeric Antigen Receptor (CAR) T cell therapies.

The anti-CD 19 binding motifs of the present disclosure may comprise antigen binding sequences as found in the antibodies described herein. In some embodiments, the anti-CD 19 binding motifs of the present disclosure comprise an antigen binding fragment provided herein.

In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise at least one HCDR provided herein, e.g., at least one HCDR disclosed in table 14. In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in table 14. In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., the three HCDRs disclosed in table 14.

In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise at least one LCDR provided herein, e.g., as disclosed in table 14. In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise two LCDRs provided herein, e.g., at least two LCDRs disclosed in table 14. In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise three LCDRs provided herein, e.g., the three LCDRs disclosed in table 14.

In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise at least one HCDR provided herein, e.g., at least one HCDR disclosed in table 14, and at least one LCDR provided herein, e.g., at least one LCDR disclosed in table 14. In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in table 14, and two LCDRs provided herein, e.g., at least two LCDRs disclosed in table 14. In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in table 14, and three LCDRs provided herein, e.g., three LCDRs disclosed in table 14.

In various embodiments, the anti-CD 19 binding motifs of the present disclosure comprise at least one heavy chain framework region (heavy chain FR) of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR chain of a heavy chain variable domain disclosed in table 14. In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in table 14. In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in table 14.

In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in table 14. In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in table 14. In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in table 14.

In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises at least one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in table 14, and at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in table 14. In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in table 14, and two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in table 14. In various embodiments, the anti-CD 19 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in table 14, and three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in table 14.

In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises one, two, or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the FRs of a corresponding heavy chain variable domain of the heavy chain variable domains disclosed in table 14. In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises one, two, or three FRs that together or each individually are at least 75% identical (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the FRs of a corresponding light chain variable domain of a light chain variable domain disclosed in table 14.

In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in table 14. In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises two heavy chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in table 14, which heavy chain variable domains may be the same or different.

In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in table 14. In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises two light chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in table 14, which light chain variable domains may be the same or different.

In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in table 14, and at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in table 14.

In various embodiments, an anti-CD 19 binding motif of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in Table 14, and two light chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in Table 14, wherein in various embodiments, (i) each heavy chain variable domain may be the same or different; or (ii) each light chain variable domain may be the same or different.

In certain embodiments, an anti-CD 19 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID No. 247. In certain embodiments, the anti-CD 19 binding motifs of the present disclosure comprise a binding motif comprising a linker according to SEQ ID NO:247 and/or a linker according to any one of SEQ ID NO: 307-313. In certain embodiments, an anti-CD 19 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 245. In certain embodiments, an anti-CD 19 binding motif of the present disclosure comprises a binding motif comprising the CSF2RA leader sequence according to SEQ ID NO: 245. In certain embodiments, the anti-CD 19 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, a linker of the present disclosure, and a leader sequence of the present disclosure. In certain embodiments, the binding motif has the sequence shown in SEQ ID NO 243. Exemplary nucleotide sequences encoding the anti-CD 19 binding motif and components thereof are found in SEQ ID NOs 244, 246, and 248. In various embodiments, the binding motif may comprise a linker according to SEQ ID NO 247 and/or a linker according to any one of SEQ ID NO 307-313, which is adjacent to one or more further linkers.

Table 14: exemplary anti-CD 19 antibody sequences (Ab11)

Antigen binding systems include, for example, bispecific and bicistronic Chimeric Antigen Receptors (CARs). The present disclosure provides, inter alia, antigen binding systems that target CD20 and a second target antigen, such as CD 19. In some embodiments, the antigen binding systems of the present disclosure comprise bispecific antigen binding systems. In some embodiments, the antigen binding systems of the present disclosure comprise a bicistronic antigen binding system (e.g., a system comprising a first CAR and a second CAR, wherein the first and second CARs are expressed in the same cell). A bicistronic CAR can comprise two CARs that bind different targets and are encoded by a single vector. A bicistronic CAR can include a first CAR comprising an anti-CD 19 binding motif and a second CAR comprising an anti-CD 20 binding motif. The binding motifs associated with the various CAR frameworks are interchangeable, and the combination of features provided in this example is exemplary and not limiting. In some embodiments, the first CAR and the second CAR of a bicistronic CAR (e.g., an anti-CD 20 CAR and an anti-CD 19 CAR) are encoded by separate genes and/or expressed as separate mRNA molecules. In some embodiments, a first CAR and a second CAR of a bicistronic CAR (e.g., an anti-CD 20 CAR and an anti-CD 19 CAR) are encoded by a single gene and/or are expressed together in a single mRNA molecule, wherein the expressed protein comprises the first CAR, a cleavable linker domain, and the second CAR. The first and second CARs of a bicistronic CAR are typically expressed together in an immune cell, e.g., a CAR-T cell, such that a single CAR-T cell expresses a CAR that targets each target antigen (e.g., each of CD20 and CD 19).

In various embodiments, the bicistronic CAR vector utilizes a ribosome skipping sequence or an internal ribosome entry site. A single vector encoding two independent CAR molecules separated by a ribosomal skip sequence can express a bicistronic CAR. In various embodiments, the bicistronic CAR comprises a first CAR and a second CAR, wherein the sequences of the first CAR and the second CAR differ only in binding motif. In various embodiments, the bicistronic CAR comprises a first CAR and a second CAR, wherein the sequences of the first CAR and the second CAR differ only in the heavy chain variable domain sequence and/or the light chain variable domain sequence. Thus, in some embodiments, the first CAR and the second CAR of a bicistronic CAR may have the same or different sequences for any or all of one or more components thereof (e.g., the same or different co-stimulatory domains). For example, one or both of the first CAR and the second CAR of a bicistronic CAR may comprise a co-stimulatory domain provided herein, such as CD28, 41BB, OX40, or ICOS co-stimulatory domain.

The CAR of the dicistronic CAR may comprise a binding motif, a hinge, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The binding motif can be an anti-CD 19 or anti-CD 20 binding motif of the present disclosure. The hinge and transmembrane domains may be the 28T (CD28) domain or the CD8K domain comprising a hinge domain and a transmembrane domain. The co-stimulatory domain may be a CD28 or 41BB co-stimulatory domain. The activation domain may be a CD3z activation domain.

In some embodiments, both the first and second binding motifs (e.g., different anti-CD 20 and anti-CD 19 binding motifs) are contained in a single bispecific CAR. In such bispecific CARs, the CAR molecule itself may be engineered to recognize more than one antigen. In tandem bispecific CARs, the first and second binding motifs are extracellular and can be characterized as a membrane proximal binding motif and a membrane distal binding motif. In some embodiments, the anti-CD 20 binding motif is membrane proximal and the anti-CD 19 binding motif is membrane distal. In other embodiments, the anti-CD 19 binding motif is distal to the membrane and the anti-CD 20 binding motif is proximal to the membrane.

Chimeric Antigen Receptors (CARs) are engineered receptors that can direct or redirect T cells (e.g., patient or donor T cells) to target a selected antigen. CARs can be engineered to recognize an antigen and upon binding to the antigen activate immune cells to attack and destroy cells bearing the antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells. CARs typically comprise an extracellular binding motif that mediates antigen binding (e.g., an anti-CD 20 and/or an anti-CD 19 binding motif), a transmembrane domain that spans or is understood to span the cell membrane when the antigen binding system is present at the cell surface or cell membrane, and an intracellular (or cytoplasmic) signaling domain.

There have been at least three "generations" of CAR compositions, according to at least one non-limiting perspective. In first generation CARs, a binding motif (e.g., single-chain fragment variable, binding motif) is linked or linked to a signaling domain (e.g., CD3 ζ) via a transmembrane domain, optionally comprising a hinge domain and one or more spacers. In second generation CARs, a costimulatory domain (CM1, such as CD28, 4-1BB or OX-40) was introduced along with a signaling domain (e.g., CD3 ζ). In third generation CARs, a second costimulatory domain (CM2) is included.

The TCR is a heterodimer consisting of an alpha chain and a beta chain. TCR signaling requires the recruitment of signaling proteins that produce immune synapses. Furthermore, the localization of the TCR at the plasma membrane depends on the CD3 complex expressed in the T cell. Engineered single chain TCRs can be generated, for example, using the transmembrane and signaling domains of the CAR construct, and methods and constructs for this are known (e.g., sTCR and TCR-CAR molecules, such as fusion of the TCR β chain with CD28 TM and CD28 and CD3 ζ). The anti-CD 20 and/or anti-CD 19 antigen binding systems of the present disclosure may comprise one or more antigen binding motifs that bind CD20 and/or CD 19. In some embodiments, the antigen binding system further comprises a co-stimulatory domain and/or an extracellular domain (e.g., a "hinge" or "spacer" region), and/or a transmembrane domain, and/or an intracellular (signaling) domain, and/or a CD3-zeta or CD 3-epsilon activation domain. In some embodiments, the anti-CD 20 and/or anti-CD 19 antigen binding systems of the present disclosure comprise at least a binding motif, a co-stimulatory domain, an extracellular domain, a transmembrane domain, and a CD3-zeta or CD3-episilon activation domain that binds human CD 20.

In some embodiments, the antigen binding system of the present disclosure may comprise an antigen binding system comprising one or more or all of a leader peptide (P), a binding motif (B), an extracellular domain of a costimulatory protein (E), a transmembrane domain (T), a costimulatory domain (C), a second costimulatory domain (C'), and an activation domain (a). In some cases, the antigen binding system is configured according to: b E T A. In some cases, the antigen binding system is configured according to: p B E T A. In some cases, the antigen binding system is configured according to: b E T C A. In some cases, the antigen binding system is configured according to: p B E T C A. In some cases, the antigen binding system is configured according to: in some cases, the antigen binding system is configured according to: p B E T C C' A. In some embodiments, the antigen binding system comprises a VH and a VL, optionally wherein the CAR is configured according to: P-VH-VL-E-T-C-A or P-VL-VH-E-T-C-A. In some embodiments, the VH and VL are linked by a linker (L), optionally wherein the CAR is configured from N-terminus to C-terminus according to: P-VH-L-VL-E-T-C-A or P-VH-L-VL-E-T-C-A.

One or more antigen binding motifs determine the target of the antigen binding system. The binding motif of the antigen binding system can comprise any binding motif, e.g., an antibody provided by the present disclosure, e.g., a binding motif of the present disclosure. In some embodiments, the binding motif may comprise an anti-CD 20 binding motif and/or an anti-CD 19 binding motif. In some embodiments, the binding motif may comprise an anti-CD 20 binding motif and/or an anti-CD 19 binding motif.

Binding motifs are used for chimeric antigen receptors at least in part because they can be engineered to be expressed as part of a single chain with other CAR components. See, e.g., U.S. Pat. Nos. 7,741,465 and 6,319,494, and Eshhar et al, Cancer immunological Immunotherapy (1997)45: 131-; finney et al, Journal of Immunology,1998,161:2791-2797, each of which is incorporated herein by reference for the binding motif domain in the CAR. A binding motif or scFv is a single chain antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain that are linked or joined together. See, e.g., U.S. Pat. Nos. 7,741,465 and 6,319,494, and Eshhar et al, Cancer Immunol Immunotherapy (1997)45:131-136, each of which is incorporated herein by reference for the binding motif domain. When derived from a parent antibody, the binding motif can retain some, all, or substantially all of the parent antibody's binding to the target antigen.

The hinge may be an extracellular domain of the antigen binding system located between the binding motif and the transmembrane domain. The hinge may also be referred to as an extracellular domain or "spacer". The hinge may contribute to receptor expression, activity and/or stability. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain. The hinge may also provide flexibility for accessing targeted antigens. The hinge comprises an immunoglobulin-like hinge domain.

In some embodiments, an antigen binding system of the present disclosure may comprise a hinge that is, is derived from, or derived from (e.g., comprises all or a fragment thereof) an immunoglobulin-like hinge domain. In some embodiments, the hinge domain is from or derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the group consisting of a hinge of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or a fragment thereof.

The hinge may be derived from natural sources or from synthetic sources. In some embodiments, the antigen binding systems of the present disclosure may comprise a hinge that is, is derived from, or derived from (e.g., comprising all or a fragment thereof) CD, CD delta, CD epsilon, CD gamma, CD8.alpha., CD8.beta., CD11 (ITGAL), CD11 (ITGAM), CD11 (ITGAX), CD11 (ITGAD), CD (ITGB), CD (B), CD (TNFRSF), CD28, CD (ITGB), CD (TNFRSF), CD (SLAMF), CD49 (ITGA), CD66 (acam), CD66 (CEACAM), CD (CLEC), CD79 (B cell antigen receptor associated complex), CD79 (beta), CD receptor associated complex (ceaca), CD150 (sldl), CD2 (slf), CD2 (tlf), CD2 (ITGA), CD1 (ITGA), CD2 (tacil), tacil (r), tacil (r (e), tacil (e), tacil (e), tacil (e), tach (e), tacil (e), tacil (e), tacil (e), tacil (e), tacil (e), tacil (e), tacil (e), tacil (e), tacil (e), tacil (e) and tacil), tacil (e) and tacil (e) for example, CD158B (KIR2 DL), CD158 (KIR3 DP), CD158 (KIRDL), CD158F (KIR2DL 5), CD158 (KIR3 DL), CD160 (BY), CD162(SELPLG), CD226 (DNAM), CD229 (SLAMF), CD244 (SLAMF), CD247 (CD-zeta), CD258(LIGHT), CD268(BAFFR), CD270 (TNFSF), CD272(BTLA), CD276 (B-H), CD279(PD-1), CD314(NKG 2), CD319 (SLAMF), CD335 (NK-p), CD336 (NK-p), CD337 (NK-p), CD352 (SLAMF), CD353 (CRAMF), CD355(CRTAM), CD357 (TNFRSF), inducible cell costimulator (ICOS), LFA-1(CD 11/NKCD), SLPG 2 (SLPG-P), SLP-76 (LAMG-P), LAMG-1, LAMG-P-1, LAMG-P, LAMG-1, LAMG-P (LAMG-1, LAMG-P), LAMG-1, LAMG-P, LAMG-1, LAMG-P (LAMG-P), LAMG-1, LAMG-P, LAMG-1, LAMD (LAM), LAM, LAMG-P, LAMD-1, LAMD-P, LAM, LAMD-P, LAMD (LAMD-P, LAMD (LAMD-P, LAM, LAMD-P, LAMD (LAMD-P, LAM, LAMD (LAM, LAMD-P, LAM, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor or Toll ligand receptor, or a fragment or combination thereof. In certain embodiments, the CAR does not comprise a CD28 hinge.

In some embodiments, an antigen binding system of the present disclosure may comprise a hinge that is, is derived from, or derived from (e.g., comprises all or a fragment thereof of) a hinge of CD8 alpha. In some embodiments, the hinge is, is from, or is derived from a CD 28. In some embodiments, the hinge is, is derived from or derived from a fragment of the hinge of CD8 alpha or a fragment of the hinge of CD28, wherein the fragment is anything less than a whole. In some embodiments, a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge comprises an amino acid sequence of a CD8 alpha hinge or a CD28 hinge that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or the C-terminus, or both. Exemplary hinge sequences include those provided in Table 54 (SEQ ID NO: 261-) -269.

The polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, a polynucleotide encoding a hinge domain comprises a polynucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the hinge domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.

In general, a "transmembrane domain" (e.g., of an antigen binding system) refers to a domain that has the property of being present in a membrane when present in a molecule at the surface or membrane of a cell (e.g., spanning a portion or all of the membrane of a cell). The co-stimulatory domain of the antigen binding systems of the present disclosure may further comprise a transmembrane domain and/or an intracellular signaling domain. It is not necessary that every amino acid in the transmembrane domain be present in the membrane. For example, in some embodiments, the transmembrane domain is characterized by a designated stretch or portion of the protein that is substantially located in the membrane. Various algorithms can be used to analyze amino acid or nucleic acid sequences to predict protein subcellular localization (e.g., transmembrane localization). The programs psort (psort. org) and progress (progress. expask. org) are examples of such programs.

The type of transmembrane domain included in the antigen binding systems described herein is not limited to any type. In some embodiments, the transmembrane domain is selected for natural association with the binding motif and/or intracellular domain. In some cases, the transmembrane domain comprises one or more amino acid modifications (e.g., deletions, insertions, and/or substitutions), for example, to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex.

The transmembrane domain may be derived from natural sources or from synthetic sources. When the source is a natural source, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains may be derived from (e.g., may comprise at least one transmembrane domain thereof) the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4 alpha, CD8beta, CD4, CD11 4, CD134, CD137, TNFSFR 4, CD154, 4-1BB/CD137, activating NK cell receptors, immunoglobulin proteins, B4-H4, BAFFR, BLAME (SLAMF 4), CD100(SEMA 44, CD103, CD160 (BTAC 72), CD4, ACA 4-CD 4, CD 4-CD 4, CD 4-7-X4, CD4, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, a ligand binding to CD83, LIGHT, LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1; CDl-la/CD18), MHC class 1molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KL 635), RF-40, PAG/Cbp, NKP 1-150, SLP 1, SLP 24, SLF-150, SLAM-4, SLF 4, SLAM-24, CD4, CD-24, CD-III, CD-III, DNA-III, DNA-III, DNA, A TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or a fragment, truncation or combination thereof. In some embodiments, the transmembrane domain may be synthetic (and may, for example, comprise predominantly hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked or linked to the cytoplasmic domain. In some embodiments, a short oligopeptide or polypeptide linker (e.g., between 2 and 10 amino acids in length) can form a link between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.

The polynucleotide and polypeptide sequences of the transmembrane domains provided herein are known. In some embodiments, a polynucleotide encoding a transmembrane domain comprises a polynucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the transmembrane domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence. Optionally, a short spacer can form a link between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains that, upon binding of the target antigen to the binding motif, cause and/or mediate an intracellular signal, e.g., that activates one or more immune cell effector functions (e.g., innate immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates activation of at least one normal effector function of the immune cell. For example, the effector function of a T cell may be cytolytic activity or helper activity involving cytokine secretion. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and/or other T cell functions. The intracellular domain may comprise a signaling domain that is an activation domain. The intracellular domain may comprise a signaling domain that is a costimulatory signaling domain.

Intracellular signaling domains that can transduce a signal when an antigen binds to an immune cell are known, any of which can be included in the antigen binding systems of the present disclosure. For example, the cytoplasmic sequence of the T Cell Receptor (TCR) is known to initiate signal transduction upon TCR binding to antigen (see, e.g., Brown lie et al, Nature Rev. Immunol.13:257-269 (2013)).

In some embodiments, the signaling domain and/or the activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, e.g., Love et al, Cold Spring Harb. Perspect. biol.2: a002485 (2010); Smith-Garvin et al, Annu. Rev. Immunol.27:591-619 (2009)).

In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB/CD137, activated NK cell receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEA 4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8beta, CD96 (Tactle), CD11a, CD11B, CD 3611 11c, CD11, CD c, ACAAM 72, ACAAL 72, CD 8672, CD 72, CD alpha-IRM receptor alpha-IL c, GAMMA-IgG c, CD 14, CD 11-IL c, CD 14-IL-7, CD c, CD-IL receptor alpha-7, CD c, CD-7, CD-IL-7, CD-IL-7, CD-IL receptor alpha-7, CD-IL receptor, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand binding to CD83, LIGHT, LTBR, Ly9(CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a/CD18), MHC 1-like molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4) SLAMF6(NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof.

The CAR may comprise a costimulatory signaling domain, e.g., to increase signaling efficacy. See U.S. Pat. Nos. 7,741,465 and 6,319,494, and Krause et al and Finney et al, (supra), Song et al, Blood 119: 696-; kalos et al, Sci Transl. Med.3:95 (2011); porter et al, n.engl.j.med.365:725-33(2011) and Gross et al, annu.rev.pharmacol.toxicol.56:59-83 (2016). The signal generated by the TCR alone may not be sufficient to fully activate the T cell, while a secondary or co-stimulatory signal may increase activation. Thus, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., innate immune cell effector functions described herein). In some embodiments, a portion of such a costimulatory signaling domain may be used, so long as the portion transduces an effector functional signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences (or fragments thereof) of a T cell co-receptor. Non-limiting examples of such T cell co-receptors include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that bind to CD 83. An exemplary costimulatory protein has the amino acid sequence of a costimulatory protein naturally present on a T cell, the complete natural amino acid sequence of the costimulatory protein being found in the NCBI reference sequence: NP _ 006130.1. In certain instances, the CAR comprises a 41BB co-stimulatory domain encoded by a sequence according to SEQ ID NO:270, as shown below:

SEQ ID NO:270

AGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat

The polynucleotide and polypeptide sequences of the signaling domains provided herein are known. In some embodiments, a polynucleotide encoding a signaling domain comprises a polynucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the signaling domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.

In various embodiments, a mechanism that modulates (e.g., reduces) the activity of the antigen binding system is desirable, for example, to minimize or curtail adverse events resulting from the activity of the antigen binding system. It may also be desirable to include an inductive "on" or "accelerator" switch in the immune cells. Suitable techniques include the use of inducible caspase-9 (U.S. application 2011/0286980) or thymidine kinase prior to, after, or simultaneously with transduction of cells with the CAR constructs of the present disclosure. Additional methods of introducing suicide genes and/or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other technologies.

In accordance with the present disclosure, on-off or other types of control switching technology may be incorporated herein. These techniques may comprise the use of dimerization domains and optionally activators of such domain dimerization, such as disclosed in Wu et al, Science 2014350 (6258), which utilize the FKBP/Rapalog dimerization system in certain cells, the contents of which are incorporated herein by reference in their entirety. Additional dimerization techniques are described, for example, in Fegan et al chem. rev.2010,110,3315-3336 and U.S. patent nos. 5,830,462; 5,834,266, respectively; 5,869,337, respectively; and 6,165,787, the contents of each of which are also incorporated herein by reference with respect to dimerization techniques. Additional dimerization pairs may comprise a cyclosporine-a/cyclophilin receptor, an estrogen/estrogen receptor (optionally with tamoxifen, 4-hydroxytamoxifen, or endooxifen), a glucocorticoid/glucocorticoid receptor, a tetracycline/tetracycline receptor, and/or a vitamin D/vitamin D receptor. Further examples of dimerization techniques can be found, for example, in WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, US patent No. 8,486,693, US 2014/0171649, and US 2012/0130076, the contents of which are further incorporated herein by reference in their entirety.

In some embodiments, the antigen binding systems of the present disclosure comprise a leader peptide (also referred to herein as a "signal peptide" or "leader sequence"). In certain embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to the amino acid sequence MEWTWVFLFLLSVTAGVHS (SEQ ID NO:249), MALPVTALLLPLALLLHAARP (SEQ ID NO:250), or MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 295).

Components of the CAR can be exchanged or "swap" as equivalent components using conventional biotechnology techniques. To provide just some non-limiting and partial examples, the CARs of the present disclosure may comprise a combination of a binding motif as provided herein with a hinge as provided herein and a co-stimulatory domain as provided herein. In certain examples, a CAR of the present disclosure can comprise a leader sequence provided herein in combination with a binding motif provided herein in combination with a hinge provided herein and a co-stimulatory domain provided herein. In various embodiments, the present disclosure provides a binding motif according to any of SEQ ID NO:251-260 in combination with (e.g., fused adjacent to) a hinge according to any of SEQ ID NO:261-269, optionally further in combination with (e.g., fused adjacent to) a 41BB co-stimulatory domain according to SEQ ID NO: 270. Some non-limiting examples of which are provided in SEQ ID NO 271-290.

A dicistronic CAR may comprise a first CAR sequence and a second CAR sequence expressed as a single polypeptide comprising a cleavable linker between the first and second CARs. An exemplary cleavable linker is furin-GSG-T2A (see, e.g., Chung et al MAbs.2015Mar-Apr; 7(2): 403-. To provide but one non-limiting example of a dicistronic CAR structure, a dicistronic CAR may comprise (a) a first CAR comprising (i) a signal peptide (e.g., CSF2RA signal peptide); (ii) an anti-CD 19 light chain variable domain; (iii) a linker (e.g., G4S linker or a plurality thereof); (iv) an anti-CD 19 heavy chain variable domain; (v) spacers or hinges (e.g., CD28T spacers); (vi) a transmembrane domain (e.g., CD28 transmembrane domain); (vii) a costimulatory domain (e.g., CD28 costimulatory domain); (viii) a stimulatory domain (e.g., a CD3z stimulatory domain); (b) a cleavable linker (e.g., a furin GSG-T2A linker); and (c) a second CAR comprising (i) a signal peptide (e.g., CD8a signal peptide); (ii) an anti-CD 20 heavy chain variable domain of the present disclosure; (iii) a linker (e.g.a linker according to SEQ ID NO:247 and/or a linker according to any one of SEQ ID NO:307 and 313); (iv) an anti-CD 20 light chain variable domain of the present disclosure; (v) spacers or hinges (e.g., CD8a spacers); (vi) a transmembrane domain (e.g., CD8 transmembrane domain); (vii) a co-stimulatory domain (e.g., 41bb co-stimulatory domain); and (viii) a stimulation domain (e.g., CD3z stimulation domain). Thus, but not limited to, an exemplary anti-CD 20/anti-CD 19 dicistronic CAR can have or comprise the nucleotide and amino acid sequences shown in SEQ ID NOS: 291 and 292.

A bispecific CAR can be a single polypeptide comprising a first binding motif of the present disclosure and a second binding motif that is an anti-CD 19 binding motif. In one non-limiting exemplary embodiment, a bispecific CAR can comprise (i) a leader (e.g., CSF2RA signal peptide), (ii) an anti-CD 20 light chain variable domain of the present disclosure; (iii) a joint; (iv) an anti-CD 20 heavy chain variable domain; (v) a linker (e.g., a truncated linker); (vi) an anti-CD 19 light chain variable domain; (vii) a joint; (viii) an anti-CD 19 heavy chain variable domain; (ix) an extracellular domain (e.g., a CD28T hinge or an IgG4 hinge); (x) A transmembrane domain (e.g., CD28 transmembrane domain); (xi) An intracellular region (e.g., CD28 intracellular co-stimulatory domain and/or 41bb co-stimulatory domain); and a stimulatory domain (e.g., CD3z stimulatory domain). Thus, but not limited to, an exemplary anti-CD 20/anti-CD 19 bispecific CAR can have or comprise the nucleotide and amino acid sequences set forth in SEQ ID NO 293-306.

Various CAR sequences, components and/or frameworks are known, including but not limited to the sequences of the hinge, spacer, transmembrane domain, costimulatory domain, stimulatory domain, binding motif and their respective variants, and CARs with the desired binding and components or frameworks can be readily constructed if, for example, a heavy chain variable domain sequence or CDR sequence and a light chain variable domain sequence or CDR sequence are provided.

The present disclosure provides, inter alia, bispecific antibodies that bind CD20 and a second target antigen, e.g., CD 19. Bispecific antibodies comprise an antibody having a first binding motif that binds a first target antigen and a second binding motif that binds a second target antigen. In some embodiments, the bispecific antibody comprises an anti-CD 20 binding motif of the present disclosure and an anti-CD 19 binding motif of the present disclosure. In some embodiments, the bispecific antibody comprises an anti-CD 20 binding motif comprising an anti-CD 20 heavy chain variable domain of the present disclosure and an anti-CD 20 light chain variable domain of the present disclosure, and an anti-CD 19 binding motif comprising an anti-CD 19 heavy chain variable domain and an anti-CD 19 light chain variable domain.

The present disclosure includes conjugates in which an antibody of the present disclosure is associated with a therapeutic agent or a detectable moiety. In various embodiments, the therapeutic agent is an anti-cancer agent provided herein. In certain embodiments, provided conjugates comprise one or more detectable moieties, "labeled" with one or more such moieties. In some such embodiments, the conjugates of the present disclosure can be used in diagnostic or imaging applications, such as diagnosing or imaging cancer. Any of a variety of detectable moieties can be used in the labeled antibody conjugates described herein. Suitable detectable moieties include, but are not limited to: various ligands, radionuclides; a fluorescent dye; chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like); a bioluminescent agent; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; a paramagnetic metal ion; an enzyme; colorimetric labels (such as, for example, dyes, colloidal gold, etc.); biotin; digoxin (dioxigenin); a hapten; and proteins from which antisera or monoclonal antibodies are available.

The present disclosure includes nucleic acids encoding the anti-CD 20 binding motifs and/or anti-CD 19 binding motifs provided herein. The present disclosure encompasses nucleic acids encoding the antibodies provided herein, including but not limited to nucleic acids encoding binding motifs (e.g., anti-CD 20 binding motif and anti-CD 19 binding motif). The present disclosure encompasses nucleic acids encoding the antigen binding systems provided herein, including but not limited to nucleic acids encoding bicistronic and bispecific chimeric antigen receptors (e.g., bicistronic and bispecific chimeric antigen receptors that bind CD20 and CD 19). The nucleic acid sequence of SEQ ID NO. 2 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO. 1 and 3-11. The nucleic acid sequence of SEQ ID NO 13 includes and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 12 and 14-22. The nucleic acid sequence of SEQ ID NO. 24 contains and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO. 23 and 25-33. The nucleic acid sequence of SEQ ID NO 35 contains and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 34 and 36-44. The nucleic acid sequence of SEQ ID NO 46 includes and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 45 and 47-55. The nucleic acid sequence of SEQ ID NO. 57 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO. 56 and 58-66. The nucleic acid sequence of SEQ ID NO 68 contains and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 67 and 69-77. The nucleic acid sequence of SEQ ID NO. 79 contains and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO. 78 and 80-88. The nucleic acid sequence of SEQ ID NO. 90 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO. 89 and 91-99. The nucleic acid sequence of SEQ ID NO 101 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 100 and 102 and 110. The nucleic acid sequence of SEQ ID NO 112 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 111 and 113 and 121. The nucleic acid sequence of SEQ ID NO 123 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 122 and 124 and 132. The nucleic acid sequence of SEQ ID NO 134 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 133 and 135-143. The nucleic acid sequence of SEQ ID NO 145 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 144 and 146 and 154. The nucleic acid sequence of SEQ ID NO:156 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO:155 and 157-165. The nucleic acid sequence of SEQ ID NO 167 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 166 and 168-176. The nucleic acid sequence of SEQ ID NO:178 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO:177 and 179-187. The nucleic acid sequence of SEQ ID NO:189 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO:188 and 190-198. The nucleic acid sequence of SEQ ID NO 200 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 199 and 201 and 209. The nucleic acid sequence of SEQ ID NO 211 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 210 and 212 and 220. The present disclosure encompasses nucleic acids encoding the anti-CD 19 binding motifs provided herein. The nucleic acid sequence of SEQ ID NO 222 comprises and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 221 and 223-231. The nucleic acid sequence of SEQ ID NO 233 contains and provides exemplary nucleic acid sequences corresponding to and encoding each of SEQ ID NO 232 and 234 and 242.

The disclosure includes vectors comprising a nucleic acid of the disclosure and/or encoding a polypeptide of the disclosure. In various embodiments, the disclosure includes vectors comprising nucleic acids encoding anti-CD 20 binding motifs and/or anti-CD 19 binding motifs provided herein. In various embodiments, the disclosure includes vectors comprising nucleic acids encoding the antibodies provided herein, including, but not limited to, nucleic acids encoding binding motif molecules (e.g., anti-CD 20 binding motifs or anti-CD 19 binding motifs). In various embodiments, the disclosure includes vectors comprising nucleic acids encoding one or more of the antigen binding systems provided herein, including, but not limited to, nucleic acids encoding bicistronic or bispecific chimeric antigen receptors (e.g., bicistronic and bispecific chimeric antigen receptors that bind CD20 and CD 19).

Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an EB virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-Puro, pBABE-neo largetTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP _2.0, TtRMPVIR, pMSCV-IRES-mCheherFP, pRetrox GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The recombinant expression vector may be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector may be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λ GT10, λ GT11, λ ZapII (Stratagene), λ EMBL4 and λ NM1149 may also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, a bicistronic IRES vector (e.g., from Clontech) is used to comprise a nucleic acid encoding an antigen binding system and an inducible expression construct as described herein.

In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alphaviruses, vaccines, adenoviral, adeno-associated, herpes, and fowlpox vectors, and preferably the ability to transform immune cells (e.g., T cells) naturally or engineered.

Recombinant expression vectors can be used, for example, in Sambrook et al, Molecular Cloning, A Laboratory Manual,3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Constructs of circular or linear expression vectors can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.

The recombinant expression vector may comprise one or more marker genes, which allow for the selection of transformed or transfected hosts. Marker genes include antimicrobial resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, etc. Suitable marker genes for recombinant expression vectors include, for example, the neomycin/G418 resistance gene, puromycin resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resistance gene.

Vectors useful in the context of the present disclosure may be "naked" nucleic acid vectors (i.e., vectors having little or no proteins, sugars, and/or lipids encapsulating them), or vectors complexed with other molecules. Other molecules that may be suitably combined with the vector include, but are not limited to, viral coat, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.

Vector DNA can be introduced into cells, such as immune cells, via conventional transformation, transfection, or transduction techniques. The terms "transformation" and "transfection" encompass a variety of art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into cells, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, gene gun, nanoparticle-mediated delivery, or electroporation. Transduction comprises viral delivery of the vector to a cell, for example by a vector disclosed herein, including but not limited to retroviruses, lentiviruses, and AAV.

The present disclosure includes cells comprising, expressing, or engineered (e.g., transformed or transduced) to comprise or express at least one vector or nucleic acid of the present disclosure. In some embodiments, the method comprises transducing a cell with a vector comprising a polynucleotide encoding at least one antigen binding system. The present disclosure includes cells comprising or transformed to comprise at least one vector encoding one or more polypeptides of the disclosure. The present disclosure includes cells comprising or transformed to comprise at least one vector encoding an anti-CD 20 binding motif and/or an anti-CD 19 binding motif provided herein. The present disclosure includes cells comprising or transformed to comprise at least one vector encoding an antibody provided herein, including but not limited to a binding motif molecule (e.g., an anti-CD 20 binding motif or an anti-CD 19 binding motif). The present disclosure includes cells comprising or transformed to comprise at least one vector encoding one or more of the antigen binding systems provided herein, including but not limited to a bicistronic or bispecific chimeric antigen receptor (e.g., a bicistronic or bispecific chimeric antigen receptor that binds CD20 and CD 19). In some embodiments, the cell is co-transfected or co-transduced with two vectors, each vector encoding a different CAR, which together are a bicistronic CAR. Transfection or transduction of cells with two different vectors (which encode two different CARs together as a bicistronic CAR) can be performed simultaneously on a single cell population, can be performed simultaneously on two different cell populations, wherein each cell population is transduced with only one of the two vectors, or can be performed independently on two different cell populations, each cell population is transduced with only one of the two vectors.

The present disclosure includes cells comprising one or more polypeptides of the present disclosure. The present disclosure includes cells comprising (e.g., expressing) an anti-CD 20 binding motif and/or an anti-CD 19 binding motif provided herein. The present disclosure includes cells comprising (e.g., expressing) an antibody provided herein, which antibody comprises, but is not limited to, a binding motif (e.g., an anti-CD 20 binding motif or an anti-CD 19 binding motif). The present disclosure includes cells comprising (e.g., expressing) one or more of the antigen binding systems provided herein, including, but not limited to, bicistronic or bispecific chimeric antigen receptors (e.g., bicistronic and bispecific chimeric antigen receptors that bind CD20 and CD 19).

In other aspects, provided herein are cells comprising a polynucleotide or vector of the present disclosure. In some embodiments, the disclosure relates to a cell, e.g., an in vitro cell, comprising a polynucleotide encoding a CAR or TCR comprising one or both of the scfvs disclosed herein. In other embodiments, the disclosure relates to a cell, e.g., an in vitro cell, comprising a polypeptide encoded by a CAR or TCR comprising one or both of the scfvs disclosed herein. In some embodiments, the polypeptide comprises the following amino acid sequence, or any combination thereof.

232 (anti-CD 19 scFv light chain):

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

221 (anti-CD 19 scFv heavy chain):

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

56 (anti-CD 20 light chain):

DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

45 (anti-CD 20 heavy chain):

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSS

56 (anti-CD 20scFv 2 light chain):

155 (anti-CD 20scFv 2 heavy chain):

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSRYVWSWIRQPPGKGLEWIGEIDSSGKTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

144 (anti-CD 20scFv 3 light chain):

DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK

177 (anti-CD 20scFv 3 heavy chain):

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYAWSWIRQPPGKGLEWIGEIDHRGFTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

78 (anti-CD 20scFv 4 light chain):

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK

67 (anti-CD 20scFv 4 heavy chain):

QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS

for the polynucleotides, vectors or polypeptides of the present disclosure, any cell may be used as a host cell. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, bacteria of the family enterobacteriaceae (enterobacteriaceae) such as Escherichia (Escherichia), e.g., Escherichia coli (e.coli); enterobacteria (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia (Serratia), such as Serratia discolorations (Serratia marcescens) and Shigella (Shigella); bacillus (bacillus) such as bacillus subtilis and bacillus licheniformis (b.licheniformis); pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (p. aeruginosa); and Streptomyces (Streptomyces). In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of: t cells, B cells, Tumor Infiltrating Lymphocytes (TILs), TCR expressing cells, Natural Killer (NK) cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, blood Platelets, thymocytes, and myeloid cells. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In certain embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL), an autologous T cell, an engineered autologous T cell (eACT)^TM) Allogeneic T cells, or any combination thereof.

Chimeric antigen receptors (CARs or CAR-T) and engineered T Cell Receptors (TCR) can be readily inserted into and expressed by immune cells (e.g., T cells) to produce binding agents. In certain embodiments, the cells (e.g., immune cells such as T cells) are obtained from a donor subject. In some embodiments, the donor subject is a human patient having a cancer or tumor. In other embodiments, the donor subject is a human patient who does not have a cancer or tumor. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject.

The cells of the present disclosure can be obtained by any source known in the art. For example, T cells may be differentiated from a population of hematopoietic stem cells in vitro, or T cells may be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, the T cells may be derived from one or more T cell lines available in the art. T cells can also be obtained using any number of techniques known to those skilled in the art (e.g., FICOLL) ^TMIsolation and/or apheresis) are taken from blood units collected from a subject. In certain embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As should be appreciated, this can be accomplished, for example, by using a semi-automatic non-countercurrent centrifuge (e.g., Cobe)^TM2991 cell processor, Baxter CytoMate^TMEtc.) to use a cleaning step. In some embodiments, the washed cells are resuspended in one or more organismsCompatible buffers, or other salt solutions with or without buffers. In certain embodiments, unwanted components of the apheresis sample are removed. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. patent publication No. 2013/0287748, which is incorporated by reference herein in its entirety.

In certain embodiments, the monocytes are depleted by lysing the red blood cells (e.g., via PERCOLL)^TMGradient, isolated by using centrifugation) to isolate T cells from PBMCs. In some embodiments, a specific subpopulation of T cells, such as CD4⁺、CD8⁺、CD28⁺、CD45RA⁺And CD45RO⁺T cells may be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed against surface markers specific to the negatively selected cells. In some embodiments, cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry (which uses a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells) can be used. For example, to enrich for CD4 by negative selection ⁺Cells, monoclonal antibody mixtures typically include antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

In some embodiments, PBMCs are used directly for genetic modification of immune cells (e.g., CARs or TCRs) using methods as described herein. In certain embodiments, after PBMC isolation, T lymphocytes may be further isolated and both cytotoxic and helper T lymphocytes sorted into naive, memory and effector T cell subsets, either before or after genetic modification and/or expansion.

In some embodiments, the identification is by comparison with CD8⁺CD8 from cell surface antigens associated with each of primary, central and effector cell types of cells⁺The cells are further sorted into naive cells, central memory cells and effector cells. In some embodimentsExpression of phenotypic markers for central memory T cells included CCR7, CD3, CD28, CD45RO, CD62L and CD127 and were granzyme B negative. In some embodiments, the central memory T cell is CD8⁺、CD45RO⁺And CD62L⁺T cells. In some embodiments, effector T cells are CCR7, CD28, CD62L, and CD127 negative and are granzyme B and perforin positive. In certain embodiments, CD4 is incorporated into a pharmaceutical composition ⁺T cells were further sorted into subpopulations. For example, CD4 can be identified by identifying cell populations having cell surface antigens⁺T helper cells are sorted into naive cells, central memory cells and effector cells.

In some embodiments, immune cells, such as T cells, are genetically modified after isolation using known methods, or immune cells are activated and expanded (or, in the case of progenitor cells, differentiated) in vitro before being genetically modified. In another embodiment, an immune cell, e.g., a T cell, is genetically modified (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) with a chimeric antigen receptor described herein and then activated and/or amplified in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. patent nos. 6,905,874; 6,867,041, respectively; and 6,797,514; and PCT publication No. WO 2012/079000, the contents of which are incorporated herein by reference in their entirety. In general, such methods comprise contacting PBMCs or isolated T cells with stimulating and co-stimulating agents (e.g., anti-CD 3 and anti-CD 28 antibodies, typically adhered to beads or other surfaces) in media with appropriate cytokines (e.g., IL-2). anti-CD 3 and anti-CD 28 antibodies attached to the same beads serve as "replacement" Antigen Presenting Cells (APCs). An example is A system, a CD3/CD28 activator/stimulator system for physiologically activating human T cells. In other embodiments, the feeder cells, as well as appropriate antibodies and cytokines, are activated and stimulated using methods such as those described in U.S. patent nos. 6,040,177 and 5,827,642 and PCT publication No. WO 2012/129514 (the contents of which are incorporated herein by reference in their entirety)T cells are stimulated to proliferate.

In various aspects of the disclosure, a cell comprising, expressing, encoding, or transformed to encode a vector or polypeptide of the disclosure (e.g., an anti-CD 20 binding motif and/or an anti-CD 20/anti-CD 19 antigen binding system of the disclosure) is a binding agent. The binding agent or population of binding agents can be used as (e.g., as an active agent for) a binding agent (a composition comprising cells that can be used as a treatment, e.g., for cancer).

The disclosure further includes methods and processes for producing an antibody agent as disclosed herein, e.g., by transforming (e.g., transducing) a cell with a vector or nucleic acid of the disclosure. In some embodiments, a method or process for producing an antibody agent as disclosed herein comprises transforming (e.g., transducing) a cell with a nucleic acid encoding at least one antigen binding system provided herein (e.g., a nucleic acid present in a vector). In general, the antibody agents described herein can be produced by immune cells, such as cells that are useful or capable of being used in adoptive cell therapy. In some embodiments, the binding agent is produced by a cell type selected from the group consisting of: TIL, T-cell, CD8 ⁺Cell, CD4⁺Cells, NK-cells, gamma-delta T cells, regulatory T cells or peripheral blood mononuclear cells. "tumor infiltrating lymphocytes" or TILs refer to leukocytes that have left the bloodstream and migrated into the tumor. Lymphocytes can be divided into three groups, which include B cells, T cells, and natural killer cells. "T cell" refers to CD3⁺A cell comprising CD4⁺Helper cell, CD8⁺Cytotoxic T cells and gamma-delta T cells.

In certain embodiments, the binding agent is produced by genetically modifying (e.g., transforming) a cell, such as an immune cell, with a nucleic acid encoding an antigen binding system and/or an expression construct described herein (e.g., (i) a first recombinant expression vector comprising a nucleic acid encoding an antigen binding system and a second recombinant expression vector comprising an inducible expression construct, (ii) a single recombinant expression vector comprising both a nucleic acid encoding an antigen binding system and an inducible expression construct, or (iii) a recombinant expression vector comprising a constitutive expression construct). Recombinant expression vectors may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages.

In some embodiments, the method comprises transducing a cell with a polynucleotide encoding an antigen binding system as disclosed herein. In some embodiments, the method comprises transducing a cell with a vector comprising a polynucleotide encoding an antigen binding system.

In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject other than a patient. In exemplary aspects, a subject may undergo leukapheresis (leukapheresis), in which leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate cells of interest, e.g., T cells. These T cell isolates can be expanded and treated by methods whereby one or more CAR constructs of the disclosure can be introduced, thereby generating CAR T cells of the disclosure.

In some embodiments, the immune cell is obtained from a subject and transformed, e.g., transduced, with an inducible expression construct or a constitutive expression construct described herein, e.g., an expression vector comprising an inducible expression construct or a constitutive expression construct described herein, to obtain the binding agent. Thus, in some embodiments, the binding agent comprises autologous cells administered to the same subject from which the immune cells were obtained. In some embodiments, the immune cell is obtained from a subject and transformed, e.g., transduced, with an inducible expression construct or a constitutive expression construct described herein, e.g., an expression vector comprising an inducible expression construct or a constitutive expression construct described herein, to obtain a binding agent for allogeneic transfer to another subject.

In certain embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient having a cancer or tumor. In other embodiments, the donor subject is a human patient who does not have a cancer or tumor.

Various compositions of the present disclosure comprise engineered cell populations, which can be produced by any means. In some embodiments, the present disclosure provides a population of human cells engineered to express an antigen binding system as described herein. In some embodiments, such populations comprise a binding agent. In some embodiments, such populations comprise cultured populations. In some embodiments, such populations are cell culture populations from a single human source, which in some embodiments may receive administration of the culture populations. As disclosed herein, a binding agent can comprise any single cell or population of cells, e.g., an engineered population of cells as provided herein.

Other aspects of the disclosure relate to compositions comprising a polynucleotide described herein, a vector described herein, a polypeptide described herein, or an in vitro cell described herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and/or adjuvant. In some embodiments, the composition comprises an excipient. In one embodiment, the composition comprises a polynucleotide encoding a CAR or TCR comprising an antigen binding molecule described herein. In another embodiment, the composition comprises a CAR or TCR comprising a TCD encoded by a polynucleotide of the disclosure. In another embodiment, the composition comprises a T cell comprising a CAR or a TCR (which comprises one or both scfvs disclosed herein).

In other embodiments, the composition is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as oral administration. It is within the ability of those skilled in the art to prepare such pharmaceutically acceptable compositions. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically in the pH range of about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, the compositions are in the form of pyrogen-free, parenterally acceptable aqueous solutions comprising the compositions described herein with or without additional therapeutic agents in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the compositions described herein (with or without at least one additional therapeutic agent) are formulated as sterile isotonic solutions that are suitably preserved. In certain embodiments, the preparation involves a formulation of the desired molecule with a polymeric compound (e.g., polylactic acid or polyglycolic acid), beads, or liposomes that provides controlled or sustained release of the product, which formulation is then delivered via depot injection (depot injection). In certain embodiments, the desired molecule is introduced using an implantable drug delivery device.

In some embodiments, the present disclosure provides pharmaceutical compositions comprising and/or delivering one or more of the present disclosure, e.g., the antigen binding systems of the present disclosure, nucleic acids encoding them, and/or cells or populations thereof comprising and/or expressing them.

In some embodiments, the present disclosure provides pharmaceutical compositions comprising and/or delivering one or more cells as provided herein, e.g., a binding agent that encodes or expresses a polypeptide provided herein, e.g., an anti-CD 20/anti-CD 19 CAR (i.e., a "binding agent pharmaceutical composition"). The binding agent pharmaceutical composition may comprise one or more cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The binding agent pharmaceutical compositions of the present disclosure may be formulated for administration according to any of the embodiments set forth herein, at least one non-limiting example of which is intravenous administration. The compositions may be formulated for intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural and/or subcutaneous routes of administration. Preferably, the compositions are formulated for parenteral routes of administration. Compositions suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which, for example, render the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilising agents, thickening agents, stabilising agents and preservatives. The binding agent pharmaceutical compositions of the present disclosure may be administered in a manner suitable for the disease to be treated (or prevented).

Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using distilled water for injection as the vehicle. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride may be used as aqueous solutions for injection, optionally with suitable solubilizers, e.g., alcohols such as ethanol, and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80^TMHCO-50, and the like.

Non-limiting examples of oily liquids include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffers or sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizing agents such as benzyl alcohol or phenol, and antioxidants. The formulated injection may be packaged in a suitable ampoule.

In one embodiment, the binding agent pharmaceutical composition is substantially free of detectable levels of contaminants, such as endotoxins, mycoplasma, Replication Competent Lentiviruses (RCL), p24, VSV-G nucleic acids, HIV gag, residual anti-CD 3/anti-CD 28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, media components, vector packaging cell or plasmid components, bacteria, and fungi. In one embodiment, the bacteria is at least one selected from the group consisting of: alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and/or group A Streptococcus pyogenes.

In various embodiments, the cells provided herein (e.g., binding agents, e.g., engineered T cells or engineered NK cells)) The binding agent pharmaceutical composition may be activated and/or amplified from, and/or produced. In some embodiments, additional steps may be performed prior to administration to the subject. For example, the binding agent can be amplified in vitro after contacting (e.g., transducing or transfecting) the immune cells with an inducible expression construct or a constitutive expression construct described herein (e.g., an expression vector comprising an inducible expression construct or a constitutive expression construct) prior to administration to a subject. In vitro amplification may be performed for 1 day or more, e.g., 2 days or more, 3 days or more, 4 days or more, 6 days or more, or 8 days or more, prior to administration to a subject. In some embodiments, in vitro amplification may be performed for 21 days or less, e.g., 18 days or less, 16 days or less, 14 days or less, 10 days or less, 7 days or less, or 5 days or less. For example, in vitro amplification may be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days prior to administration to a subject. A binding agent pharmaceutical composition comprising, for example, a binding agent (e.g., an engineered T cell or an engineered NK cell) can be formulated for administration at a desired dose, e.g., 10 ⁴To 10⁹One cell/kg body weight (e.g., 10)⁵To 10⁶Individual cells/kg body weight). Certain embodiments of the present disclosure include methods of administering to a subject a pharmaceutical composition as described herein, such as, for example, a binding agent described herein (e.g., an engineered cell population of the present disclosure), a protein therapeutic described herein, a composition comprising a binding agent, and/or a composition comprising a protein therapeutic, e.g., in an amount effective to treat a subject when administered in an appropriate dosing regimen.

In some embodiments, the binding agent is autologous to the subject, and prior to isolation of the immune cells from the subject, the subject may be immunologically naive, immune, diseased, or in another condition. In some embodiments, the binding agent can be stimulated with an antigen (e.g., a TCR antigen) during in vitro amplification. Antigen-stimulated expansion may optionally be supplemented with expansion under conditions that non-specifically stimulate lymphocyte proliferation, such as, for example, anti-CD 3 antibody, anti-Tac antibody, anti-CD 28 antibody, or Phytohemagglutinin (PHA). The amplified binding agent may be administered directly to the subject or may be frozen for future use, i.e., for subsequent administration to the subject.

In some embodiments, the binding agent is treated with interleukin-2 (IL-2) ex vivo prior to infusion into the cancer patient, and the cancer patient is treated with IL-2 after infusion. Furthermore, in some embodiments, the cancer patient may undergo preliminary lymph depletion (lymphodepletion) -temporary ablation of the immune system prior to administration of the binding agent. The combination of IL-2 treatment and preparatory lymph depletion may enhance the persistence of the binding agent. In some embodiments, the binding agent is transduced or transfected with a nucleic acid encoding a cytokine, which can be engineered to provide constitutive, regulatable, or time-controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that act to enhance T lymphocyte survival during the systolic phase, which can promote the formation and survival of memory T lymphocytes.

In certain embodiments, the binding agent is administered prior to, substantially simultaneously with, or subsequent to the administration of another therapeutic agent, such as a cancer therapeutic agent. The cancer therapeutic agent can be, for example, a chemotherapeutic agent, a biologic agent, or radiation therapy. In some embodiments, the subject receiving the binding agent is not administered a treatment sufficient to cause immune cell depletion, such as lymphodepleting chemotherapy or radiotherapy.

In some embodiments, the dosage administered to a subject may vary with the embodiment, the composition employed, the method of administration, and the site and subject being treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician may determine a therapeutically effective amount of a composition to be administered to a human or other subject to treat or prevent a medical condition. The precise amount of the composition required for therapeutic effectiveness may depend on a variety of factors such as, for example, the activity of the binding agent and the route of administration.

A suitable number of binding agent cells can be administered to the subject. Although the individual binding agent cells described herein are capable of expanding and providing therapeutic benefit, in some embodiments, administration 10 is administered²Or more, e.g. 10³One or more, 10⁴One or more,10⁵One or more, or 10⁸One or more binding agent cells. In some embodiments, 10 is administered to a subject¹²Or less, e.g. 10¹¹Or less, 10⁹Or less, 10⁷Or less, or 10⁵One or less of the binding agent cells described herein. In some embodiments, administering 10 described herein²-10⁵、10⁴-10⁷、10³-10⁹Or 10⁵-10¹⁰A plurality of binder cells. Can be administered, e.g. 10⁴To 10 ⁹One cell/kg body weight (e.g., 10)⁵To 10⁶Individual cells/kg body weight) of a dosage of the binding agent pharmaceutical composition. May be at, for example, about 2X10⁶Individual cell/kg, about 3X10⁶Individual cell/kg, about 4X10⁶Individual cell/kg, about 5X10⁶Individual cell/kg, about 6X10⁶Individual cell/kg, about 7X10⁶Individual cell/kg, about 8X10⁶Individual cell/kg, about 9X10⁶Individual cell/kg, about 1X10⁷Individual cell/kg, about 2X10⁷Individual cell/kg, about 3X10⁷Individual cell/kg, about 4X10⁷Individual cell/kg, about 5X10⁷Individual cell/kg, about 6X10⁷Individual cell/kg, about 7X10⁷Individual cell/kg, about 8X10⁷Individual cell/kg or about 9X10⁷The binding agent pharmaceutical composition is administered at a dose of individual cells/kg.

The dosage of the binding agent described herein may be administered to the mammal once or in a series of sub-doses over a suitable period of time, for example once per day, half-week, weekly, bi-weekly, half-month, bi-month, half-year or annually as desired. Dosage units comprising an effective amount of the binding agent can be administered in a single daily dose, or the total daily dose can be administered in two, three, four or more divided doses administered daily, as desired.

The practitioner may select the appropriate mode of administration. The route of administration may be parenteral, for example by injection, nasal, pulmonary or transdermal administration. Systemic or local administration can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the composition is selected for parenteral delivery, inhalation, or delivery through the digestive tract, such as oral administration. The dose and method of administration may vary depending on the weight, age, condition, etc. of the subject, and may be appropriately selected.

In various embodiments, the binding agents described herein can be incorporated into a pharmaceutical composition. Pharmaceutical compositions comprising the binding agents of the present disclosure may be formulated by known methods (as described in Remington's Pharmaceutical Sciences,17th edition, ed. alfonso r. gennaro, Mack Publishing Company, Easton, Pa. (1985)). In various instances, a pharmaceutical composition comprising a binding agent of the present disclosure can be formulated to comprise a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Compositions comprising a binding agent of the present disclosure may comprise a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt.

In various embodiments, compositions comprising a binding agent as described herein, e.g., sterile formulations for injection, can be formulated according to conventional pharmaceutical practice using distilled water for injection as the vehicle. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride may be used as aqueous solutions for injection, optionally with suitable solubilizers, e.g., alcohols such as ethanol, and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 ^TMHCO-50, and the like. As disclosed herein, the pharmaceutical composition comprising the binding agent may be in any form. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.

The choice or use of any modality may depend in part on the intended mode of administration and therapeutic application. For example, a composition comprising a binding agent of the present disclosure intended for systemic or local delivery may be in the form of an injectable or infusible solution. Thus, compositions comprising the binding agents of the present disclosure can be formulated for administration by parenteral modes (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). Parenteral administration refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subconjunctival, subarachnoid, intraspinal, epidural, intracerebral, intracranial, cervical and intrasternal injection and infusion.

The route of administration may be parenteral, for example by injection, nasal, pulmonary or transdermal administration. Systemic or local administration can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.

In various embodiments, pharmaceutical compositions comprising the binding agents of the present disclosure may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating a composition comprising the binding agent of the present disclosure in the required amount in a suitable solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating a composition comprising the binding agents of the present disclosure into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying which yields a powder of the composition containing the binding agent of the present disclosure plus any additional desired ingredient from a previously sterile-filtered solution thereof (see below). Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersants and by the use of surfactants. Prolonged absorption of injectable compositions comprising a binding agent of the present disclosure can be achieved by including in the compositions comprising a binding agent of the present disclosure an agent that delays absorption, such as monostearate salts and gelatin.

Pharmaceutical compositions comprising a binding agent of the present disclosure may be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions comprising the binding agents of the present disclosure may be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, binders, and then mixing in unit dosage forms as required by generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is that amount which provides a suitable dosage within the specified range. Non-limiting examples of oily liquids include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffers or sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizing agents such as benzyl alcohol or phenol, and antioxidants. The formulated injection may be packaged in a suitable ampoule.

In some embodiments, compositions comprising the binding agents of the present disclosure can be formulated for storage at temperatures below 0 ℃ (e.g., -20 ℃ or-80 ℃). In some embodiments, compositions comprising a binding agent of the present disclosure can be formulated for storage at 2-8 ℃ (e.g., 4 ℃) for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 11/2 years, or 2 years). Thus, in some embodiments, compositions comprising a binding agent of the present disclosure are stable for storage at 2-8 ℃ (e.g., 4 ℃) for at least 1 year.

In some cases, a pharmaceutical composition comprising a binding agent of the present disclosure can be formulated as a solution. In some embodiments, compositions comprising a binding agent of the present disclosure can be formulated, for example, as a buffer solution at a suitable concentration and suitable for storage at 2-8 ℃ (e.g., 4 ℃). Pharmaceutical compositions comprising a binding agent as described herein may be formulated in an immunoliposome composition. Liposomes with extended circulation time are disclosed, for example, in U.S. Pat. No. 5,013,556.

In certain embodiments, compositions comprising the binding agents of the present disclosure may be formulated with a carrier that protects the composition from rapid release, such as a controlled release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods of preparing such formulations are known. See, e.g., J.R. Robinson (1978) "suspended and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

In some embodiments, compositions comprising an agent of the present disclosure can be formulated into compositions suitable for intrapulmonary administration (e.g., via an inhaler or nebulizer) to a mammal, such as a human. Methods for formulating such compositions are known. Dry powder inhaler formulations and suitable systems for formulation administration are also known. Pulmonary administration can be oral and/or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, Dry Powder Inhalers (DPIs) and nebulizers. For example, a composition comprising an agent of the present disclosure can be administered to the lungs of a subject by a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are known and include, but are not limited to: (AstraZeneca；London,England)Inhaler (Cambridge,Mass.)；(GlaxoSmithKline; London, England); and ECLIPSE^TM(Sanofi-Aventis; Paris, France). See also, for example, PCT publications WO 04/026380, WO 04/024156, and WO 01/78693. DPI devices have been used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, a composition comprising an agent of the present disclosure can be administered intrapulmonary by a metered dose inhaler. These inhalers rely on a propellant to deliver discrete doses of the molecule to the lung. Additional devices and methods of intrapulmonary administration are set forth, for example, in U.S. patent application publication nos. 20050271660 and 20090110679, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, compositions comprising a binding agent of the present disclosure may be formulated for delivery to the eye, e.g., in the form of a pharmaceutically acceptable solution, suspension, or ointment. Formulations for treating the eye may be in the form of sterile aqueous solutions containing, for example, additional ingredients such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants and stabilizing agents, nonionic wetting or clarifying agents, and viscosity increasing agents. The formulations described herein may be administered topically to the eye of a subject in need of treatment (e.g., a subject with AMD) by conventional methods, e.g., in the form of drops containing one or more compositions, or by bathing the eye in a therapeutic solution.

In certain embodiments, various devices for introducing a drug into the vitreous cavity of the eye may be suitable for administering compositions comprising the binding agents of the present disclosure. For example, U.S. publication No. 2002/0026176 describes a drug-containing plug that can be inserted through the sclera so that it protrudes into the vitreous cavity to deliver a medicament into the vitreous cavity. In another example, U.S. patent No. 5,443,505 describes an implantable device for introduction into the suprachoroidal space or avascular region for sustained release of a drug to the interior of the eye. U.S. Pat. nos. 5,773,019 and 6,001,386 each disclose an implantable drug delivery device attachable to the scleral surface of the eye. Additional methods and devices for delivering therapeutic agents to the Eye (e.g., transscleral patches and via contact lens delivery) are described, for example, in Ambati and Adams (2002) Prog Retin Eye Res 21(2): 145-151; ranta and Urtti (2006) Adv Drug Delivery Rev 58(11): 1164-1181; barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1):1-10 (10); gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45: 2342-; kim et al (2007) Ophthalmic Res 39: 244-254; and PCT publication No. WO 04/073551, the disclosure of which is incorporated herein by reference in its entirety.

In various embodiments, subcutaneous administration may be accomplished by devices such as syringes, prefilled syringes, auto-injectors (e.g., disposable or reusable), pen injectors, patch injectors, wearable injectors, dynamic syringe infusion pumps with subcutaneous infusion sets, or other devices used for subcutaneous injections in combination with a binder drug.

The injection system of the present disclosure may employ a delivery pen as described in U.S. patent No. 5,308,341. Pen-type devices are commonly used to self-deliver insulin to patients with diabetes. Such devices may comprise at least one injection needle (e.g., a 31-gauge needle of about 5 to 8mm in length), which is typically pre-filled with one or more therapeutic unit doses of a therapeutic solution, and which can be used to rapidly deliver the solution to a subject with as little pain as possible. A drug delivery pen contains a vial holder in which vials of therapeutic agent or other drug may be housed. The pen may be a completely mechanical device, or it may be combined with electronic circuitry to accurately set and/or indicate the dose of medicament injected into the user. See, for example, U.S. patent No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the kit contains one or more disposable replacement needles. Pen devices suitable for delivering any one of the presently characterized compositions comprising the binding agents of the present disclosure are also described, for example, in U.S. patent nos. 6,277,099; 6,200,296, respectively; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. Microneedle-based pen devices are described, for example, in U.S. patent No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also Scandinavian Health Ltd, a Precision Pen Injector (PPI) device MOLLY ^TM。

In some embodiments, a composition comprising a binding agent of the present disclosure may be delivered to a subject by means of topical administration that is independent of the transport of the binding agent to its intended target tissue or site via the vascular system. For example, a composition comprising a binding agent of the present disclosure can be delivered by injection or implantation of a composition comprising a binding agent of the present disclosure or by injection or implantation of a device containing a composition comprising a binding agent of the present disclosure. In certain embodiments, upon topical administration near a target tissue or site, a composition comprising a binding agent of the present disclosure or one or more components thereof may diffuse to the intended target tissue or site that is not the site of administration.

In some embodiments, a composition comprising a binding agent of the present disclosure can be administered topically to a joint, such as directly to the joint (e.g., into the joint space) or near the joint. Examples of intra-articular joints to which compositions comprising the binding agents of the present disclosure may be topically administered include, for example, the hip, knee, elbow, wrist (wrist), sternoclav, temporomandibular, wrist (carpal), tarsal, ankle and any other joint that has an arthritic condition. Compositions comprising the binding agents of the present disclosure may also be administered to the bursa, such as, for example, the acromion, the biceps radius, the ulna cubalis, the deltoid, infrapatella, the ischia, and any other bursa.

In some embodiments, the compositions provided herein comprising a binding agent of the present disclosure are presented in unit dosage forms, which may be suitable for self-administration. Such unit dosage forms may be provided in a container, typically, for example, a vial, cartridge, pre-filled syringe or disposable pen. A dosator, such as the dosator arrangement described in us patent No. 6,302,855, may also be used, for example, with the injection system described herein.

The pharmaceutical solution can comprise a therapeutically effective amount of a composition comprising a binding agent of the present disclosure. Such effective amounts can be readily determined based in part on the effect of the administered composition comprising a binding agent of the present disclosure, or the combined effect of the composition comprising a binding agent of the present disclosure and one or more additional active agents (if more than one agent is used). A therapeutically effective amount of a composition comprising a binding agent of the present disclosure may also vary depending on factors such as the disease state, age, sex, and weight of the individual and the ability of the composition (and one or more additional active ingredients) to elicit a desired response in the individual, e.g., to improve at least one condition parameter, e.g., to improve at least one symptom of a complement-mediated disorder. For example, a therapeutically effective amount of a composition comprising a binding agent of the present disclosure can inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent the disorder and/or any symptom of the disorder. A therapeutically effective amount is also a therapeutically effective amount wherein the therapeutically beneficial effect outweighs any toxic or detrimental effects of a composition comprising a binding agent of the present disclosure.

Compositions comprising a binding agent of the present disclosure may be administered as a fixed dose or in milligrams per kilogram (mg/kg) dose. In some embodiments, the dosage may also be selected to reduce or avoid the production of antibodies or other host immune responses against one or more antigen binding molecules in a composition comprising a binding agent of the present disclosure. Although in no way intended to be limiting, exemplary doses of a binding agent, such as a composition comprising a binding agent of the present disclosure, comprise, for example, 1-1000mg/kg, 1-100mg/kg, 0.5-50mg/kg, 0.1-100mg/kg, 0.5-25mg/kg, 1-20mg/kg, and 1-10 mg/kg. Exemplary doses of compositions comprising a binding agent of the present disclosure include, but are not limited to, 0.1mg/kg, 0.5mg/kg, 1.0mg/kg, 2.0mg/kg, 4mg/kg, 8mg/kg, or 20 mg/kg.

Suitable human dosages of any composition comprising a binding agent of the present disclosure can be further evaluated in, for example, a phase I dose escalation study. See, e.g., van Gurp et al (2008) Am J transfer 8(8): 1711-1718; hanouska et al, (2007) Clin Cancer Res 13(2, part 1): 523-531; and Hetherington et al (2006) analytical Agents and Chemotherapy 50(10) 3499-3500.

In various embodiments, a pharmaceutical composition can comprise a nucleic acid of the present disclosure, e.g., a vector. Methods of formulating Pharmaceutical compositions comprising nucleic acids of the present disclosure are known, for example, in the books of the Series of Drugs and the Pharmaceutical Sciences: a Series of Textbooks and monograms (Dekker, N.Y.). For example, a solution or suspension for parenteral, intradermal, or subcutaneous application may comprise the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid and the like; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in, for example, ampoules, disposable syringes or vials of more than one dose made of glass or plastic.

Pharmaceutical compositions suitable for injection comprising a nucleic acid of the present disclosure may comprise sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers may comprise physiological saline, bacteriostatic water, Cremophor el.tm. (BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Sterility, stability, viscosity and other factors associated with effective therapeutic use may be considered. For example, one method of maintaining fluidity includes the use of coatings such as lecithin, the maintenance of the desired particle size, and the use of surfactants. In some cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. In some cases, prolonged absorption of an injectable composition comprising a nucleic acid of the present disclosure can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating one or more ingredients such as a combination of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like) and/or by filter sterilization. In some cases, dispersions are prepared by incorporating the active molecule into a sterile vehicle that contains a basic dispersion medium and an antibacterial or antifungal agent. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying from previously sterile-filtered solutions thereof.

Oral compositions comprising a nucleic acid of the present disclosure may comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the nucleic acids of the present disclosure can be combined with excipients and used in the form of, for example, tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions comprising the nucleic acids of the present disclosure can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any one of the following ingredients or molecules of similar properties: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipient such as starch or lactose, disintegrant such as alginic acid,Or corn starch; lubricating agents, e.g. magnesium stearate orGlidants, such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

In some embodiments, the nucleic acid may be administered by any method suitable for administration of nucleic acid agents (e.g., DNA vaccines). These methods include, for example, gene guns, bio-injectors and skin patches as well as needle-free methods, such as the microparticle DNA vaccine technology disclosed in U.S. patent No. 6,194,389, and transdermal needle-free vaccination of mammals using vaccines in powder form, such as disclosed in U.S. patent No. 6,168,587. Furthermore, intranasal delivery is possible as described in Hamajima et al, (1998) clin. Liposomes (e.g., as described in U.S. patent No. 6,472,375). In some cases, microencapsulation may be used. In addition, biodegradable targeted microparticle delivery systems can be used (e.g., as described in U.S. patent No. 6,471,996).

Compositions comprising a nucleic acid of the present disclosure may comprise components such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, mixtures thereof or any component for inclusion in a therapeutic composition comprising a nucleic acid. The nucleic acid composition can comprise, for example, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, phosphate buffered saline, water, and emulsions, such as oil/water or water/oil emulsions, and various types of wetting agents compatible with pharmaceutical administration. Complementary active molecules can also be incorporated into compositions of the present disclosure comprising a nucleic acid of the present disclosure. Compositions of the present disclosure comprising a nucleic acid of the present disclosure may comprise a stabilizer and a preservative, as well as any of the carriers described herein, optionally with the proviso that they are acceptable for use in vivo. See Martin REMINGTON 'S PHARM. SCI.,15th Ed. (Mack Publ. Co., Easton (1975) and Williams & Williams, (1995) and "PHYSICIAN' S DESK REFERENCE,"52nd ed., Medical Economics, Montvale, N.J. (1998).

The methods described herein include making and using pharmaceutical compositions comprising nucleic acids of the disclosure. Pharmaceutical compositions comprising a nucleic acid of the present disclosure are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intracranial, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

The nucleic acid composition may comprise a buffer or a pH adjusting agent. The buffer may be a salt prepared from an organic acid or base. The buffers of the present disclosure comprise organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris hydroxymethyl aminomethane hydrochloride, and phosphate buffers. Additional carriers include polymeric excipients or additives such as polyvinylpyrrolidone, polysucrose (a polymeric sugar), dextran (e.g., cyclodextrin, such as 2-hydroxypropyl-ortho-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweetening agents, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as TWEEN, for example)And TWEEN) Lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelators (e.g., EDTA).

In certain embodiments, wherein the nucleic acid of the present disclosure is a vector, the present disclosure comprises a composition for gene transduction and/or gene therapy, e.g., a composition comprising a viral particle, e.g., an AAV particle and/or a retrovirus, such as a lentiviral particle. As used in reference to viral titer, the term "genomic particle (gp)" or "genomic equivalent" refers to the number of virions containing a recombinant viral genome (e.g., a recombinant AAV DNA genome), regardless of infectivity or functionality. The number of genomic particles in the carrier formulation can be measured. As used in reference to viral titer, the terms "Infectious Unit (IU)", "infectious particle", or "replication unit" refer to the number of infectious and replication competent recombinant viruses, e.g., the number of recombinant AAV vector particles, as can be measured by an infection center assay (also referred to as a replication center assay). As used in reference to viral titers, the term "Transduction Unit (TU)" refers to the number of infectious recombinant vector particles (e.g., recombinant AAV vector particles) that result in the production of a functional transgene product.

In some embodiments, the composition comprises, e.g., 2x10 per dose⁶To 2x10¹²、2x10⁷To 2x10¹¹Or 2x10⁸To 2x10¹¹A viral particle comprising DNA. In certain embodiments, the concentration or titer of the vector in a unit dosage form is, for example, at least: (a)1x10¹²particle/mL, 2X10¹²particle/mL, 3X10¹²particle/mL, 4X10¹²particle/mL, 5X10¹²particle/mL, 6X10¹²particle/mL, 7X10¹²particle/mL, 8X10¹²particle/mL, 9X10¹²particle/mL, 10X10¹²particle/mL, 15X10¹²particle/mL, 20X10¹²particle/mL, 25X10¹²particle/mL, or 50X10¹²particles/mL;(b)1x10⁹TU/mL、2x10⁹TU/mL、3x10⁹TU/mL、4x10⁹TU/mL、5x10⁹TU/mL、6x10⁹TU/mL、7x10⁹TU/mL、8x10⁹TU/mL、9x10⁹TU/mL、10x10⁹TU/mL、15x10⁹TU/mL、20x10⁹TU/mL, 25 or 50x10⁹TU/mL; or (c)1x10¹⁰IU/mL、2x10¹⁰IU/mL、3x10¹⁰IU/mL、4x10¹⁰IU/mL、5x10¹⁰IU/mL、6x10¹⁰IU/mL、7x10¹⁰IU/mL、8x10¹⁰IU/mL、9x10¹⁰IU/mL、10x10¹⁰IU/mL、15x10¹⁰IU/mL、20x10¹⁰IU/mL、25x10¹⁰IU/mL or 50X10¹⁰IU/mL. Such embodiments do not limit the unit dosages encompassed by the present disclosure and do not limit the various measures of dosages that can be used in conjunction with the various compositions of the present disclosure comprising the nucleic acids of the present disclosure. For example, for vector genomes per kilogram of subject (Vg/Kg) or Vg/dose, particle dose, concentration or amount can be measured and/or expressed. The preferred manner of measuring and/or expressing the particle dose, concentration or amount may vary depending on various factors, such as the route of administration.

The present disclosure provides techniques for simultaneously targeting CD20 and another antigen (e.g., CD 19). In some embodiments, the present disclosure provides techniques for initiating and/or modulating an immune response. In some embodiments, the present disclosure provides techniques for treating cancer (e.g., cancer characterized by cells having surface-expressed CD 20).

The present specification encompasses the use of the binding agent pharmaceutical compositions provided herein for the treatment or prevention of cancer. Another aspect of the disclosure relates to a method of treating or preventing a malignant tumor, comprising administering to a subject in need thereof an effective amount of a binding agent pharmaceutical composition, e.g., wherein the cells comprise at least one antigen binding system provided herein. The methods of the present disclosure comprising administering a pharmaceutically effective amount of a binding agent pharmaceutical composition of the present disclosure can be used to treat cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate a tumor from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain embodiments, the methods provided herein induce a complete response. In some embodiments, the methods provided herein induce a partial response. In certain embodiments, the binding agent pharmaceutical composition is, comprises as an active agent, or comprises as the only active agent a cell provided herein, e.g., a cell comprising or expressing at least one CAR of the present disclosure. In some embodiments, the binder pharmaceutical composition comprises a bicistronic CAR system comprising an anti-CD 20 CAR and an anti-CD 19 CAR, or the binder pharmaceutical composition comprises a bispecific anti-C20/anti-CD 19 CAR of the present disclosure.

In various embodiments, the disclosure includes use of a binding agent pharmaceutical composition provided herein to induce or provide immunity to a cancer in a subject. The present disclosure further comprises methods of preventing cancer in a subject by administering to the subject a binding agent pharmaceutical composition provided herein. The present disclosure further comprises methods of inducing an immune response in a subject by administering to the subject a binding agent pharmaceutical composition provided herein. In certain embodiments, the binding agent pharmaceutical composition is, comprises as an active agent, or comprises as the only active agent a cell provided herein, e.g., a cell comprising or expressing at least one CAR of the present disclosure. In some embodiments, the binder pharmaceutical composition comprises a bicistronic CAR system comprising an anti-CD 20 CAR and an anti-CD 19 CAR, or the binder pharmaceutical composition comprises a bispecific anti-C20/anti-CD 19 CAR of the present disclosure.

In certain embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject a polynucleotide, vector, antibody, or antigen binding system disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding an antigen binding system or an antibody (e.g., an antigen binding system). In another embodiment, the method comprises administering a vector comprising a polynucleotide encoding an antigen binding system or an antibody. In another embodiment, the method comprises administering to the subject an antigen binding system or an antibody.

Another aspect of the disclosure relates to a method of making a cell expressing a CAR or TCR comprising transducing a cell with a polynucleotide disclosed herein under suitable conditions. In some embodiments, the method comprises transducing a cell with a polynucleotide encoding a CAR or a TCR, as disclosed herein. In some embodiments, the method comprises transducing a cell with a vector comprising a polynucleotide encoding a CAR or a TCR. In certain embodiments, the present disclosure provides a T cell therapy wherein the binding agent pharmaceutical composition comprises a T cell transfected or transduced with a vector comprising a polynucleotide sequence encoding an antigen binding agent of the disclosure (e.g., an antigen binding system). In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject that is not a patient. In one embodiment, the T cell therapy of the present disclosure is autologous cell therapy (eACT)^TM). According to this embodiment, the method may include collecting blood cells from the patient. Isolated blood cells (e.g., T cells) can then be engineered to express the antigen binding systems of the present disclosure. In certain embodiments, the binding agent is administered to the patient. In some embodiments, the binding agent treats or is intended to treat cancer in the patient. For example, in one embodiment, the binding agent reduces the size of the tumor. In various embodiments, the cells of the present disclosure may be freshly isolated cells from a human subject, freshly isolated cells from a cell culture, or cells that have been stored, e.g., frozen.

Another aspect of the disclosure relates to a method of inducing immunity to a tumor comprising administering to a subject an effective amount of a cell comprising a polynucleotide described herein, a vector described herein, or a CAR or TCR described herein. In one embodiment, the method comprises administering to a subject an effective amount of a cell comprising a polynucleotide encoding a CAR or TCR disclosed herein. In another embodiment, the method comprises administering to the subject an effective amount of a cell comprising a vector comprising a polynucleotide encoding a CAR or TCR disclosed herein. In another embodiment, the method comprises administering to the subject an effective amount of a cell comprising a CAR or a TCR encoded by a polynucleotide disclosed herein.

Another aspect of the present disclosure relates to a method of inducing an immune response in a subject comprising administering an effective amount of an engineered immune cell of the present application. In some embodiments, the immune response is a T cell mediated immune response. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cell comprises a CAR or TCR, wherein the CAR or TCR comprises a THD as described in the present disclosure. In some embodiments, the target cell is a tumor cell.

Another aspect of the disclosure relates to a method for treating or preventing a malignant tumor, the method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one CAR or TCR, and wherein the CAR or TCR comprises one or both of the scfvs disclosed herein.

Another aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a polynucleotide, vector, CAR or TCR, cell or composition disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a vector comprising a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a CAR or a TCR encoded by a polynucleotide disclosed herein. In another embodiment, the method comprises administering a cell comprising a polynucleotide encoding a CAR or a TCR or a vector comprising a polynucleotide encoding a CAR or a TCR.

In some embodiments, the method of treating cancer in a subject in need thereof comprises T cell therapy. In one embodiment, the T cell therapy of the present disclosure is an engineered autologous cell therapy (eACT) ^TM). According to this embodiment, the method may comprise collecting blood from the patientA cell. The isolated blood cells (e.g., T cells) can then be engineered to express the CARs or TCRs of the disclosure. In a specific embodiment, the CAR T cells or TCR T cells are administered to a patient. In some embodiments, the CAR T cells or TCR T cells treat a tumor or cancer in the patient. In one embodiment, the CAR T cells or TCR T cells reduce the size of the tumor or cancer.

In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject other than a patient.

The T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10⁴At least about 10 cells⁵At least about 10 cells⁶At least about 10 cells⁷At least about 10 cells⁸At least about 10 cells⁹Individual cell or at least about 10¹⁰And (4) cells. In another embodiment, the therapeutically effective amount of T cells is about 10⁴One cell, about 10⁵One cell, about 10⁶One cell, about 10⁷One cell or about 10 ⁸And (4) cells. In a specific embodiment, the therapeutically effective amount of the CAR T cell or TCR T cell is about 2X10⁶Individual cell/kg, about 3X10⁶Individual cell/kg, about 4X10⁶Individual cell/kg, about 5X10⁶Individual cell/kg, about 6X10⁶Individual cell/kg, about 7X10⁶Individual cell/kg, about 8X10⁶Individual cell/kg, about 9X10⁶Individual cell/kg, about 1X10⁷Individual cell/kg, about 2X10⁷Individual cell/kg, about 3X10⁷Individual cell/kg, about 4X10⁷Individual cell/kg, about 5X10⁷Individual cell/kg, about 6X10⁷Individual cell/kg, about 7X10⁷Individual cell/kg, about 8X10⁷Individual cell/kg, or about 9X10⁷Individual cells/kg.

The methods of the present disclosure can be used to treat cancer, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof in a subject. In certain embodiments, the method induces a complete response. In other embodiments, the method elicits a partial response.

In certain embodiments, the cancer comprises cells expressing CD19, such as cells expressing CD19 on the cell surface. In certain embodiments, the cancer comprises cells expressing CD20, such as cells expressing CD20 on the cell surface. In certain embodiments, the cancer comprises cells that each independently express both CD19 and CD20, e.g., express on the surface of the cells.

Cancers that may be treated include tumors that are not vascularized, have not substantially vascularized, or have vascularized. Cancer may also include solid or non-solid tumors. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is a cancer of leukocytes. In other embodiments, the cancer is a cancer of plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or myeloma. In certain embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), Acute Lymphocytic Leukemia (ALL) and Hemophagocytic Lymphohistiocytosis (HLH), B-cell prolymphocytic leukemia, B-cell acute lymphocytic leukemia ("BALL"), blastic plasmacytoid dendritic cell tumor, burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloid Leukemia (CML), chronic or acute granulomatous disease, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), hairy cell leukemia, hemophagic syndrome (macrophage activating syndrome (MAS), hodgkin's disease, large cell granuloma, large cell granulomatosis, acute lymphoblastic leukemia (DLBCL), lymphoblastic syndrome (MAS), lymphoblastic disease, lymphomatosis, lymphomas, lymphomatosis, lymphosis, lymphomatosis, lymphosis, lymphomatosis, lymphoblastosis, lymphosis, lymphoblastosis, lymphosis, and lymphosis, and the like, lymphosis, and the like, Leukocyte adhesion deficiency, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, Monoclonal Gammopathy of Unknown Significance (MGUS), multiple myeloma, myelodysplasia, and myelodysplastic syndrome (MDS), myeloid diseases including, but not limited to, Acute Myeloid Leukemia (AML), non-Hodgkin's lymphoma (NHL), plasmacytoid diseases (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmacytoma (e.g., plasmacytoma; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (Crohn-deep-Monuk syndrome; Takatsi disease; PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), Small or large cell follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphocytic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or waldenstrom's macroglobulinemia, or a combination thereof. In one embodiment, the cancer is myeloma. In a specific embodiment, the cancer is multiple myeloma. In another embodiment, the cancer is leukemia. In one embodiment, the cancer is acute myeloid leukemia.

In various instances, the method of treating cancer using the binding agent pharmaceutical compositions provided herein is autologous cell therapy. In various instances, the method of treating cancer using the binding agent pharmaceutical compositions provided herein is allogeneic cell therapy.

Certain methods of using the binding agent pharmaceutical compositions provided herein include collecting blood cells from a subject. Isolated subject blood cells (e.g., T cells) can then be engineered to express, for example, the antigen binding systems of the present disclosure. In some embodiments, the binding agent is administered to the subject. In some embodiments, the binding agent treats cancer in the subject. In one embodiment, the binding agent reduces the size of the tumor.

In various embodiments, the cell therapies provided herein for use in the present disclosure can be administered to a subject in the course of a treatment that further comprises administration of one or more additional therapeutic agents or therapies that are not the cell therapies provided herein. In certain embodiments, the present disclosure provides combination therapies for the treatment of cancer comprising administering an anti-cancer agent to a subject receiving and/or in need of a binding agent provided herein.

In certain embodiments, the binding agents provided herein can be administered to a subject who has previously received, is scheduled to receive, or is in the course of a treatment regimen that includes additional anti-cancer therapies. In various embodiments, the additional agent or therapy administered in combination with a binding agent provided herein as described herein can be administered simultaneously with a binding agent provided herein, on the same day as a binding agent provided herein, or on the same week as a binding agent provided herein. In various embodiments, the additional agent or therapy administered as described herein in combination with a binding agent provided herein can be administered such that administration of the binding agent provided herein and administration of the additional agent and therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of the binding agent provided herein. In various embodiments, the frequency of administration of one or more additional agents can be the same as, similar to, or different from the frequency of administration of the binding agents provided herein.

The agents or therapies used in combination with a binding agent provided herein can be administered as a single therapeutic composition or dose with a binding agent provided herein, as a separate composition administered concurrently with a binding agent provided herein, or administered in a temporally different manner than the administration of a binding agent provided herein. When a binding agent provided herein is used in combination with an additional agent, the binding agent provided herein can be co-formulated with the additional agent or the binding agent provided herein can be formulated separately from the additional agent formulation.

In some embodiments, the method further comprises administering a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent of choice is a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens and related beneficial biomarkers are described in U.S. provisional patent applications 62/262,143 and 62/167,750, which are incorporated herein by reference in their entirety. These describe, for example, methods of conditioning a patient in need of a T cell therapy comprising administering to the patient a prescribed beneficial dose of cyclophosphamide (200 mg/m)²Day-2000 mg/m²Day) and prescriptionsAmount of fludarabine (20 mg/m)²Day-900 mg/m²Day). One such dosage regimen involves treating the patient, comprising administering to the patient about 500mg/m per day²Cyclophosphamide per day and about 60mg/m²Fludarabine/day for 3 days, and then administering a therapeutically effective amount of the engineered T cells to the patient. In other embodiments, the antigen binding molecule, transduced (or otherwise engineered) cells (e.g., CARs or TCRs), and chemotherapeutic agent are each administered in an amount effective to treat the disease or condition in the subject.

In certain embodiments, a composition comprising an immune effector cell expressing a CAR and/or a TCR disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cycloxan) ^TM) (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetylomelamine; nitrogen mustards (nitrosgen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), ramustine (ranimustine); antibiotics, such as aclar Examples of the active ingredient include, but are not limited to, streptomycin (acrinomycin), actinomycin (actinomycin), anthranomycin (anthracycline), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cacinomycin), calicheamicin (calicheamicin), carbamicin (carminomycin), carcinosin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorelbumin (detorubicin), 6-diaza-5-oxo-L-norvaline (DON), doxorubicin (doxobicin), epirubicin (epirubicin), esorubicin (esubicin), idarubicin (idarubicin), sisomicin (myomycin), mycins (mitomycins), mycins (actinomycin), streptomycins (mucomycin), streptomycins (gentamycin), and streptomycins (streptomycins), and streptomycins (flavomycins), and a pharmaceutically acceptable carrier, or a pharmaceutically acceptable carrier, a carrier, a, Streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), azinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate); purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); amsacrine (amsa) crine); tabularil (bestrabucil); bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); podophyllinic acid (podophyllic acid); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);razoxane (rizoxane); sisofilan (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin; cytarabine (arabine) ("Ara-C"); cyclophosphamide; thiotepa (thiotepa); taxols (taxoids), such as paclitaxel (paclitaxel) (TAXOL) ^TMBristol-Myers Squibb) and docetaxel (doxetaxel) ((R)Rhone-Poulenc Rorer); chlorambucil (chlorambucil); gemcitabine; 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide (ifosfamide); mitomycin C; mitoxantrone (mitoxantrone); vincristine; vinorelbine; navelbine (navelbine); oncostatin (novantrone); teniposide (teniposide); daunomycin (daunomycin); aminopterin (aminopterin); xeloda; ibandronate (ibandronate); CPT-11; rubbing devicePameose inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid derivatives, e.g. Targretin^TM(bexarotene), Panretin^TM(alitretinoin); ONTAK^TM(denileukindeftitox); esperamicin (esperamicin); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some embodiments, a composition comprising a CAR and/or TCR-expressing immune effector cell may be administered in combination with an anti-hormonal agent that acts to modulate or inhibit the effect of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), aromatase-inhibiting 4(5) -imidazole, 4-hydroxytamoxifene, trioxifene (trioxifene), woloxifene (keoxifene), LY117018, onapristone (onapristone), and toremifene (toremifene) (Fareston); and antiandrogens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide), and goserelin (goserelin); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Combinations of chemotherapeutic agents are also administered where appropriate, including but not limited to CHOP, i.e., cyclophosphamide Doxorubicin (hydroxydoxorubicin), vincristineAnd prednisone.

In some embodiments, the chemotherapeutic agent is administered simultaneously with or within a week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

A variety of additional therapeutic agents may be used withThe compositions described herein are used in combination. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumabPembrolizumab (pembrolizumab)Pembrolizumab, abaclizumab (pidilizumab) (CureTech), and alezumab (Atezolizumab) (Roche). Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to, ibrutinib (ibrutinib)Oxamumumab (ofatumumab)Rituximab (rituximab)Bevacizumab (bevacizumab)Trastuzumab (trastuzumab)trastuzumab emtansineImatinib (imatinib) Cetuximab (cetuximab)Panitumumab (panitumumab)Cetuximab (Catumaxomab), ibritumomab tiuxetan(ibritumomab), ofatumumab, tositumomab (tositumomab), benitumomab (brentuximab), alemtuzumab (alemtuzumab), gemtuzumab (gemtuzumab), erlotinib (erlotinib), gefitinib (gefitinib), vandetanib (vandetanib), afatinib (afatinib), lapatinib (lapatinib), neratinib (neratinib), axitinib (axitinib), masitinib (masitinib), pazopanib (papaniib), sunitinib (sunitinib), sorafenib (sorafenib), toceranib, lestatinib (lestatinib), axitinib (axitinib), ceritinib (cedaniib), sorafenib (valacinib), sunitinib (sorafenib), sunitinib (netatinib), sunitinib (neratinib), sunitinib (sorafenib), sunitinib (neritinib), sunitinib (sorafenib), sunitinib (sorafenib), sunitinib (sorafenib), sunitinib (sorafenib), sunitinib, Dasatinib (dasatinib), nilotinib (nilotinib), panatinib (ponatinib), ridatinib (raditinib), bosutinib (bosutinib), lestatinib (lestauutinib), ruxolitinib (ruxolitinib), palitinib (pacritinib), cobimetinib (cobimetinib), semetinib (selumetinib), trametinib (trametiniib), benimetinib (palitinib), arertinib (aletinib), ceritinib (ceritinib), crizotinib (crotinib), aflibercept (aflibercept), addipertide (adottide), dinierein (geleintinbitrix), mTOR inhibitors such as everolitis (evolinib), and sirolimus inhibitors such as oviridine inhibitors (vegiviroc), sirolimus (sirolimus) such as paclobulin (CDK), and sirolimus (sirolimus) such as (sirolimus) inhibitors (sirolimus), and (sirolimus) such as (sirolimus) inhibitors such as (sirolimus (clotrimminthib).

In additional embodiments, a composition comprising an immune cell comprising a CAR and/or a TCR is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, triamcinolone, and combinations thereof)mcinolone)), non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin (aspirin), ibuprofen (ibuprofen), naproxen (naproxen), methotrexate, sulfasalazine, leflunomide (leflunomide), anti-TNF drugs, cyclophosphamide, and mycophenolate (mycophenolate). Exemplary NSAIDs include ibuprofen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include paracetamol (acetaminophen), oxycodone (oxycodone), tramadol, or propoxyphene hydrochloride (tramadol of prolyphene hydrochloride). Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists, (e.g., etanercept) Adalimumab (adalimumab)And infliximab (infliximab)Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecule. Exemplary DMARDs include azathioprine (azathioprine), cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline (minocycline).

In certain embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, "cytokine" means a protein released by one cell population that acts on another cell as an intercellular mediator. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. The cell factor includes growth hormone, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; pine needleRelaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); hepatic Growth Factor (HGF); fibroblast Growth Factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substances (mullerian-inhibiting substance); mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve Growth Factor (NGF) such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon-alpha, beta and-gamma; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines. In various embodiments, the binding agents provided herein for use in the present disclosure can be administered to a subject in a course of treatment that further includes administration of an anti-inflammatory agent. Anti-inflammatory agents may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include tramadol, acetaminophen, oxycodone, propoxyphene hydrochloride. Display device Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept)Adalimumab (adalimumab)And infliximab (infliximab)Chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecule. Exemplary DMARDs include azathioprine (azathioprine), cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline (minocycline).

In various embodiments, the binding agents provided herein for use in the present disclosure can be administered to a subject in a course of treatment that further includes administration of CHOP. CHOP consists of: (C) cyclophosphamide, an alkylating agent that destroys DNA by binding to it and causing the formation of cross-links; (H) hydroxydaunorubicin (also known as doxorubicin or adriamycin), an intercalator that disrupts DNA by inserting itself between DNA bases; (O) vincristine (vincristine), which prevents cell replication by binding to the protein tubulin; and (P) prednisone (prednisone) or (P) prednisolone (prednisone), which is a corticosteroid.

Further exemplary embodiments. The present disclosure includes, but is not limited to, the following exemplary embodiments:

embodiment 1. an isolated polynucleotide encoding a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR) comprising (i) an antigen binding molecule, (ii) a costimulatory domain, and (iii) an activation domain, wherein the costimulatory domain comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen binding molecule consisting essentially of or consisting of: (i) an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence of any one of the constructs set forth in SEQ ID NOs 232, 221, 56, 45, 155, 144, 177, 78 and 67.

Embodiment 2. the polynucleotide of embodiment 1, wherein the transmembrane domain is a transmembrane domain of: 4-1BB/CD137, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, CD3epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of a T cell receptor, or any combination thereof.

Embodiment 3. the polynucleotide of embodiment 1 or 2, wherein the intracellular domain comprises the signaling regions of: 4-1BB/CD137, activating NK cell receptor, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD29, CD3, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD8alpha, CD8beta, CD f (Tactile), CDl la, CDl lb, CDl, CDL, CDS, CECDM f, CRT, cytokine receptor, CD-10, DAP f (GAMMET 226), GAMMET receptor, GAITEM 14, GAITAG 72, GAITAG f, GAITAG-IgG f, GAITAG-like IgG f, GAITAG f, GAITAG-like IgG f, GAITAG, LFA-1, a ligand that specifically binds to CD83, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1(CDl la/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF 1; CD 150; IPO-3), SLAMF4(CD 244; 2B4), AMF6 (NTB-A; Lyl 356), SLAMF7, SLP-76, TNF receptor protein, TNFR2, VLNCR, VLNCE receptor, SLLL receptor ligand, SLLL-08, or a combination thereof.

Embodiment 4 the polynucleotide of embodiments 1 to 3, wherein at least one of said antigen binding molecules specifically binds to an antigen selected from the group consisting of: 5T4, an alpha-fetoprotein, a B Cell Maturation Antigen (BCMA), CA-125, a carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD56, CD123, CD138, C-Met, CSPG4, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, bound HER1-HER2, bound HER2-HER3, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, IL-llRalpha, kappa chain, lambda chain, melanoma-related antigen, mesothelin, MUC-1, mutated p53, mutated prostate specific antigen, RORAR 53, or VEGFR2, or a combination thereof.

Embodiment 5 the polynucleotide of any one of embodiments 1 to 4, wherein said polynucleotide encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NOs 232, 221, 56, 45, 155, 144, 177, 78 and 67.

Embodiment 6. polypeptides encoded by the polynucleotides of any one of embodiments 1 to 5.

Embodiment 7. a polypeptide comprising the amino acid sequence shown in SEQ ID nos. 232, 221, 56, 45, 155, 144, 177, 78 and 67.

Embodiment 8. a vector comprising the polynucleotide of any one of embodiments 1 to 5.

Embodiment 9 the vector of embodiment 7, wherein said vector is an adenoviral vector, an adeno-associated vector, a DNA vector, a lentiviral vector, a plasmid, a retroviral vector or an RNA vector, or any combination thereof.

Embodiment 10. a cell comprising the polynucleotide of any one of embodiments 1 to 5, the polypeptide of claim 6 or 7, the vector of claim 8 or 9, or any combination thereof.

Embodiment 11 a composition comprising the polynucleotide of any one of embodiments 1 to 5, the polypeptide of claim 6 or 7, the vector of claim 8 or 9, the cell of claim 10, or any combination thereof.

Embodiment 12. a method of making a cell comprising the polynucleotide of any one of claims 1 to 5, the polypeptide of embodiment 6 or 7, the vector of claim 8 or 9, or any combination thereof.

Embodiment 13. a method of inducing immunity against a tumor comprising administering to a subject an effective amount of the polynucleotide of any one of claims 1 to 5, the polypeptide of embodiment 6 or 7, the vector of embodiment 8 or 9, the cell of embodiment 10, the composition of embodiment 11, or any combination thereof.

Embodiment 14 use of the polynucleotide of any one of embodiments 1 to 5, the polypeptide of embodiment 6 or 7, the vector of embodiment 8 or 9, the cell of embodiment 11, or the composition of embodiment 12 for the manufacture of a medicament for treating cancer in a subject in need thereof.

Embodiment 15 the use of embodiment 15, wherein the cancer is Acute Lymphoblastic Leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell tumor, burkitt lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), hairy cell leukemia, hodgkin's disease, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, Monoclonal Gammopathy of Unknown Significance (MGUS), multiple myeloma, myelodysplasia, and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), a plasma cell proliferative disease (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), plasmacytoma (including plasma cell cachexia; isolated myeloma; isolated plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytomas), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small-or large-cell follicular lymphoma, Splenic Marginal Zone Lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphocytic leukemia ("TALL"), T-cell lymphoma, transformative follicular lymphoma, or Walsh macroglobulinemia, or a combination thereof.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. Any definitions or terms provided in any references that are incorporated by reference, to the extent that they are different from the terms and discussions provided herein, control over the terms and definitions of the present invention. The contents of all references cited throughout this application are expressly incorporated herein by reference.

Examples

Example 1

The present example provides exemplary generated anti-CD 20 heavy and light chain variable domains and combinations thereof. CDR sequences of exemplary anti-CD 20 heavy chain variable domains and anti-CD 20 light chain variable domains, and thus exemplary combinations of HCDR and LCDR, are also provided below in tables 4-13. Tables 4-13 comprise exemplary nucleic acid sequences encoding exemplary variable domains (and thus exemplary nucleic acid sequences encoding the identified CDRs of the exemplary variable domains are also provided).

To determine cellular binding of exemplary heavy and light chain variable domains of the disclosure, cellular binding of purified IgG at a concentration of 10nM was characterized by flow cytometry. Antibodies to CHO-S cells overexpressing CD20, Raji and Namalwa CD20+ cell lines; and incubation with EoL-1 and CHO-S CD 20-cell lines. FITC-LC was used for IgG detection. The ratio of binding of each antibody to the negative control was calculated. Exemplary cell binding of selected anti-CD 20 antibodies is provided in table 15.

Watch 15

Example 2

This example provides a bicistronic and bispecific CAR. Bicistronic and bispecific CARs comprise two binding motifs (located in two CAR molecules or on a single CAR molecule, respectively). The first binding motif binds to CD20, and the second binding motif binds to CD 19. Antibody sequences that bind CD19 and/or antibody sequences that can be used to construct binding motifs, antibodies, and antigen binding systems that bind CD19 are known. This example uses the antibody sequence of an anti-CD 19 binding agent, referred to herein as antibody Ab11, as shown in table 14.

The bispecific CAR of this example was generated to comprise the following domains: a first binding motif, a second binding motif, a hinge, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. In this example, each bispecific CAR included a first binding motif comprising a heavy chain variable domain and a light chain variable domain from a single set of exemplary antibody sequences of example 1 (i.e., both from the same table in tables 4-13, corresponding to Ab1-Ab 10). Thus, a binding motif can be identified by reference to a source antibody or source table, and refers to a binding motif having a heavy chain variable domain and a light chain variable domain of a source antibody as listed in the respective table. In this example, each bispecific CAR included a second binding motif comprising a heavy chain variable domain and a light chain variable domain that were the heavy chain variable domain and the light chain variable domain of antibody Ab11 (i.e., SEQ ID NOS: 221 and 232). The bispecific CAR of this example comprises a 28T (CD28) domain comprising a hinge domain and a transmembrane domain. The bispecific CAR of this example comprises a CD28 co-stimulatory domain. The bispecific CAR of this example comprises a CD3z activation domain.

This example includes four dicistronic CARs, identified as Bic-2, Bic-8, Bic-9, and Bic-14. Each of the four dicistronic CARs included a first CAR construct comprising an anti-CD 20 binding motif comprising the heavy and light chain variable domain pair of example 1, as shown in table 16 below, and a second CAR construct comprising an anti-CD 19 binding motif.

Table 16: anti-CD 20 binding motif sequence for bicistronic CAR

CAR	Binding motifs	VH SEQ ID	VL SEQ ID
				Bic-2	Ab3	45	56
Bic-8	Ab8	155	166
				Bic-9	Ab9	177	188
Bic-14	Ab4	67	78

Example 3

Isolation of CD4 by Positive selection from apheresis Material from healthy donors⁺And CD8⁺T cells, and are used to generate anti-CD 20 monovalent or anti-CD 20/anti-CD 19 bicistronic CAR T cell products. T cells were activated with bound anti-CD 3 and soluble CD28 antibodies and transduced with lentiviral vectors encoding CAR constructs. As a control, non-transduced (NTD) T cells were generated in parallel from the same donor T cells. On the day of harvest (8-10 days of manufacture), CAR T cell products were stained and analyzed by flow cytometry to assess transduction efficiency and for co-culture assays. The transduction efficiency of T cells with a monovalent CAR-encoding vector and a bicistronic CAR-encoding vector is monitored.

To determine the T cell transduction efficiency of vectors encoding monovalent CARs, CAR-T products were stained with a panel of antibodies (anti-CD 3, anti-CD 4, anti-CD 8, and anti-linker antibodies) in the presence of a fixable viability dye and analyzed by flow cytometry to assess the percentage of CAR-positive cells that survived (see WO/US2017/041534, which is incorporated herein by reference for anti-linker antibodies). An anti-linker antibody is an antibody that binds to the linker between the heavy and light chains of the anti-CD 20 CAR binding motif and is used to measure transduction efficiency. Controls included non-transduced cells (NTD), cells transduced with retrovirus containing control anti-CD 19 binding agent, and cells transduced with control anti-CD 20 binding agent (Ab12 binding motif).

To determine the T cell transduction efficiency of the bicistronic CAR-encoding vectors, CAR-T products were stained with a panel of antibodies (including anti-CD 3, anti-CD 4, anti-CD 8, anti-idiotype, and anti-linker antibodies) in the presence of a fixable viability dye and analyzed by flow cytometry to assess the percentage of CAR-positive cells that survived. The anti-idiotype antibody binds to the binding motif of Ab11 anti-CD 19 binding motif. Thus, the anti-idiotype antibody binds to an anti-CD 19 CAR. It was used to measure the transduction efficiency of anti-CD 19 CARs. Anti-linker antibodies were used to measure transduction efficiency of anti-CD 20 CARs. Controls included non-transduced cells (NTD), cells transduced with retroviruses containing control anti-CD 19 binding agents, and cells transduced with control anti-CD 20/anti-CD 19 bispecific CARs (Ab13/Ab14 bispecific; Ab11/Ab12 bispecific).

Table 17A: transduction efficiency of anti-CD 20 monovalent CARs

Binding motif #	Transduction efficiency (% CD20 CAR +)
		Ab3	65.6
Ab5	60.9
		Ab6	70.7
Ab10	51.1
		Ab7	52.8
Ab8	40.6
		Ab9	33
Ab1	44.4
		Ab4	62.6
Ab2	35.8
		NTD	0.25
Ab11	47.8
		Ab12	60.7

Table 17B: transduction efficiency of anti-CD 20/anti-CD 19 bicistronic CAR

Example 4

This example provides, inter alia, an exemplary method of co-culturing CAR-T cells with cells expressing a CAR-T target antigen. To facilitate tracking of T cells in culture, CellTrace was used according to the manufacturer's instructions ^TMThe Violet (CTV) reagent labels CAR-T cells, which are then washed with R-10% medium. To facilitate tracking of cells expressing the CAR-T target antigen ("target cells"), the target cells are engineered to express luciferase. Luciferase-expressing target cells include Nalm6 and Raji, both of which express both CD19 and CD20 antigens. In addition, prepareNalm6 and Raji cells expressing CD19 or CD20 (knock-out cells, or KO). These CD19KO and CD20KO cells were selected from Nalm6 and Raji parental cell clones and expressed CD20 but not CD19, or CD19 but not CD20, respectively. CD19KO and CD20KO strains were generated and used as controls to functionally evaluate the antigen binding of each CAR of bicistronic anti-CD 20/anti-CD 19 CAR-expressing cells.

Luciferase-expressing target cells were plated in R-10% medium (day 0 of co-culture) with CTV-labeled CAR-T cells at different rates. This ratio can be referred to as the ratio of effector (CAR-T) cells to target cells (effector: target or E: T). To plate cells at the desired ratio, CAR-T cells were serially diluted 2 to 3 fold while keeping the number of target cells per well constant at 25,000 cells. The co-cultures were incubated at 37 ℃ for 16 hours (h) or 4 days and function assessed as described below.

Example 5

In this example, T cells were co-cultured with target cells as described in example 4. T cell mediated cytotoxicity was measured as a function of the decrease in target luciferase signal in co-culture wells compared to the signal emitted by target cells plated alone. On day 4 after the start of co-culture, D-luciferin substrate was added to the co-culture wells at a final concentration of 0.14mg/mL, and the plates were incubated at 37 ℃ for 10 minutes in the dark. In Varioskan^TMLUX orAnd reading the luminous signal in the Flash multi-mode microplate reader. T cell mediated cytotoxicity was calculated as follows: % cytotoxicity ═ luciferase signal of [ 1- (sample of interest/target-alone control ]]*100。

Controls included non-transduced (NTD) T cells (i.e., T cells that did not express the CAR), cells transduced with retroviruses containing control anti-CD 19 binding agents, cells transduced with control anti-CD 20 binding agents, cells transduced with control anti-CD 20/anti-CD 19 bispecific CARs (Ab13/Ab14 bispecific; Ab15/Ab16 bispecific) as negative controls.

Table 18: percentage cytotoxicity against CD20 CAR-T in coculture after 4 days (Nalm6 wild-type cells)

Table 19: percentage cytotoxicity against CD20 CAR-T in coculture after 4 days (Raji wild-type cells)

Table 20: percentage cytotoxicity of anti-CD 20/anti-CD 19 bicistronic CAR-T in coculture after 4 days (Nalm6 wild type cells)

Table 21: percentage cytotoxicity in coculture after 4 days of anti-CD 20/anti-CD 19 bicistronic CAR-T (Raji wild type cells)

Table 22: percentage cytotoxicity of anti-CD 20/anti-CD 19 bicistronic CAR-T in coculture after 4 days (Nalm6 CD19KO cells)

Table 23: percentage cytotoxicity of anti-CD 20/anti-CD 19 bicistronic CAR-T in coculture after 4 days (Nalm6 CD20KO cells)

Table 24: percentage cytotoxicity in coculture after 4 days of anti-CD 20/anti-CD 19 bicistronic CAR-T (Raji CD19KO cells)

Table 25: percentage cytotoxicity in coculture after 4 days of anti-CD 20/anti-CD 19 bicistronic CAR-T (Raji CD20KO cells)

Example 6

In this example, T cells were co-cultured with target cells as described in example 4. After 16 hours of co-culture, supernatants were collected and analyzed for cytokine levels using the Meso Scale Discovery V-PLEX pro-inflammatory group 1 human kit according to the manufacturer's instructions. Supernatants from co-cultures of T cell products plated at a 1: 1E: T ratio with antigen expressing target cells were analyzed for levels of interferon gamma (IFN-. gamma.), IL-2, tumor necrosis factor alpha (TNF-. alpha.), and IL-10 secretion mediated by antigen conjugation. All samples were diluted to within the detection range. The level of each cytokine is reported in pg/mL, and the lower and upper limit of quantitation for each assay is reported.

Controls included non-transduced (NTD) T cells (i.e., T cells that did not express the CAR), cells transduced with retroviruses containing control anti-CD 19 binding agents, cells transduced with control anti-CD 20 binding agents, cells transduced with a control bispecific antigen binding system (Ab13/Ab14 bispecific), and cells transduced with a bispecific antigen binding system (Ab15/Ab16 bispecific) as negative controls.

Table 26: cytokine production (pg/mL) in 16 hour coculture of anti-CD 20 CAR-T with Nalm6 wild type cells

Table 27: cytokine production (pg/mL) in 16 hour coculture of anti-CD 20 CAR-T with Raji wild type cells

Table 28: cytokine production (pg/mL) in 16 hour coculture of anti-CD 20/anti-CD 19 bicistronic CAR-T with Nalm6 wild type cells

Table 29: cytokine production (pg/mL) in 16 hour coculture of anti-CD 20/anti-CD 19 bicistronic CAR-T with Raji wild type cells

Table 30: cytokine production (pg/mL) in 16 hour coculture of anti-CD 20/anti-CD 19 bicistronic CAR-T with Nalm6 CD19KO cells

Table 31: cytokine production (pg/mL) in 16 hour coculture of anti-CD 20/anti-CD 19 bicistronic CAR-T with Nalm6 CD20KO cells

Example 7

T cells were co-cultured with target cells as described in example 4. After 16 hours of co-culture, T cell products plated with antigen positive target cells at a specific E: T ratio were harvested, stained with a panel of antibody fluorophores to identify T cells (CD3, CD4, CD8) and 4-1BB (an activation marker), and analyzed by flow cytometry. The fixable viability dye allows analysis of surviving cells. Events were in live cells (vital dye negative), lymphocytes (using forward scatter [ FSC ]]Regio-and-sidescatter [ SSC]Region map), single cells (using FSC region and FSC height map), then systematically gated on T cells (CD3 +).The level of activation of T cells (i.e., 4-1 BB) is then analyzed⁺Percentage of cells); the 4-1BB gating threshold was set based on the level of NTD control T cell expression.

Controls included non-transduced (NTD) T cells (i.e., T cells that did not express the CAR) as negative controls, cells transduced with retroviruses containing control anti-CD 19 binding agents, cells transduced with control anti-CD 20 binding agents, cells transduced with a control bispecific antigen binding system (Ab13/Ab14 bispecific), and cells transduced with a bispecific antigen binding system.

Table 32: activation of anti-CD 20 CAR-T cells in 16 hour coculture with Nalm6 wild-type and Raji wild-type cells

Table 33: activation of anti-CD 20/anti-CD 19 bicistronic CAR-T cells in 16 hour coculture with Nalm6 wild type, Nalm6 CD20KO, and Nalm6 CD19KO cells

Example 8

T cells were co-cultured with target cells as described in example 4. The proliferative capacity of the T cell product was determined by flow cytometry analysis of cell division-driven dilution of CTV dye in response to antigen-expressing target cells, compared to NTD control T cells. On day 4 after the start of co-culture, T cell products plated at a 3:1E: T ratio with target cells expressing antigen were harvested, stained with a panel of antibody fluorophores (CD3, CD4, CD8) in the presence of a fixable viability dye to identify viable T cells and analyzed by flow cytometry. The percentage of proliferating cells and the Mean Fluorescence Intensity (MFI) of the CTV signal are reported. The decrease in MFI of CTV is proportional to the number of rounds of cell division experienced by the product (i.e. the lower the MFI of CTV, the more proliferation the cells experience).

Controls included non-transduced (NTD) T cells (i.e., T cells that did not express the CAR), cells transduced with retroviruses containing control anti-CD 19 binding agents, cells transduced with control anti-CD 20 binding agents, cells transduced with a control bispecific antigen binding system (Ab13/Ab14 bispecific), and cells transduced with an Ab11/Ab12 bispecific antigen binding system as negative controls.

Table 34: fluorescent anti-CD 20 CAR-T cells in four-day coculture with Nalm6 wild-type and Raji wild-type cells

Table 34: percentage proliferation of anti-CD 20 CAR-T cells in four-day coculture with Nalm6 wild-type and Raji wild-type cells

		Nalm6 WT	Raji WT
				Binding motif #	Ab#	% proliferation	% proliferation
2	Ab3	68.77	74.95
				3	Ab5	81.21	78.35
5	Ab6	40.43	78.68
				6	Ab10	52.31	87.53
7	Ab7	76.23	78.48
				8	Ab8	76.6	84.79
9	Ab9	75.91	87.42
				10	Ab1	33.94	53.61
14	Ab4	76.1	80.6
				16	Ab2	38.16	85.05
NTD	N/A	37.38	48.15
				Ab11	N/A	74.96	86.98
Ab12	N/A	86.32	72.9

Table 35: fluorescent anti-CD 20/anti-CD 19 bicistronic CAR-T cells in four-day co-culture with Nalm6 wild-type, Nalm6 CD19KO, Raji wild-type and Raji CD19KO cells

Table 36: percentage proliferation of anti-CD 20/anti-CD 19 bicistronic CAR-T cells in four-day coculture with Nalm6 wild-type, Nalm6 CD19KO, Raji wild-type and Raji CD19KO cells

Example 9

This example provides data relating to hinges for the anti-CD 20 antigen binding system. The CAR T cells of this example comprise a monovalent anti-CD 20 CAR. This example tests CAR-T constructs against a disseminating luciferase-expressing Raji human B cell lymphoma model in NSG mice. Blood samples were monitored weekly for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. Whole blood was drawn for analysis on days 7, 13, 20, 27, 34 and 41. BLI was performed on days 5, 12, 19, 26, 33, and 47. The study was terminated on day 55.

CAR-T cells of this study are shown in table 37 below, where each of the CAR-T cell types of table 37 was administered to six mice. Table 37 provides the experimental conditions or groups mentioned in the subsequent tables of this example. All CAR-T cells were administered intravenously at a dose of 2E +07 cells/mL (QDx 1). All mice also received a 36. mu.g dose of human IL-2((Q12Hx2) QDx 3). Human IL-2 promotes the persistence and survival of CAR-T. The constructs of CAR T cells of groups 4, 5 and 7-10 of table 37 comprise a 41BB co-stimulatory domain. Group 6 CAR T cells of table 37 comprise a CD28 co-stimulatory domain.

Table 37:

mice were studied to be well-tolerated by all CAR-T cell therapies. Within each group, a number of indicators were monitored including weight change associated with treatment, mortality associated with treatment, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased longevity. The results are shown in table 38 below, noting that the data reflects monitoring by terminating the experiment on day 55.

Watch 38

Example 10

This example provides data related to the anti-tumor efficacy of CAR-T cells comprising a dicistronic CAR comprising an anti-CD 19 CAR and an anti-CD 20 CAR, the anti-CD 20 CAR comprising various anti-CD 20 binding motifs and/or hinges.

Bicistronic anti-CD 20/anti-CD 19 CAR-T cells were tested against disseminating luciferase-expressing Raji CD19KO B cell lymphoma in NSG mice. Blood samples were monitored weekly for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. Whole blood was drawn for analysis on days 7, 13, 20, 27, 34 and 41. BLI was performed on days 5, 12, 19, 26, 33, 40 and 48. The study was terminated on day 55. The CAR-T cells of this study, as shown in lines 5-14 of table 39 below, were bicistronic CAR-T cells comprising an anti-CD 20 CAR and an anti-CD 19 CAR (Ab 11 CAR comprising a 28T (CD28) hinge and a CD28 co-stimulatory domain) as shown in table 39. Table 39 provides the experimental conditions or groups mentioned in the subsequent tables of this example. CAR-T cells were administered to six mice (except four mice received non-transduced T cells (row 2 of table 39)). All CAR-T cells were administered intravenously at a dose of 1E +07 cells/mL (rows 2-7, 9, 11, and 13) or 4E +06 (rows 8, 10, 12, and 14) (QDx 1). All mice also received a 36. mu.g dose of human IL-2((Q12Hx2) QDx 3).

Watch 39

A number of indicators were monitored including treatment-related weight change, treatment-related mortality, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased lifespan (% ILS). The results are shown in table 40 below, noting that the data reflects monitoring by terminating the experiment on day 55.

Mice were studied to be well-tolerated by all CAR-T cell therapies. All CAR-T cell treatments tested elicited an anti-tumor response. All treatment regimens resulted in a median tumor growth delay of >30.2 days and an Increased Lifespan (ILS) > 226.7%. All treatment regimens resulted in a median% T/C of 0% on day 19, except for treatment with non-transduced (NTD) cells (24%).

Watch 40

Example 11

This example provides data relating to the anti-tumor efficacy of CAR-T cells comprising a bispecific CAR comprising an anti-CD 19 binding motif and an anti-CD 20 binding motif. Bispecific anti-CD 20/anti-CD 19 CAR-T cells were tested against disseminating luciferase-expressing Raji B cell lymphoma tumors in NSG mice. Blood samples were monitored weekly for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. Whole blood was drawn for analysis on days 7, 13, 20, 27, 32 and 41. BLI was performed on days 5, 12, 19, 26, 33, 40 and 47. The study was terminated on day 54.

As shown in table 41, lines 4-8 below, the CAR-T cells of this study were bispecific CAR-T cells comprising an anti-CD 19 binding motif and an anti-CD 20 binding motif, wherein the anti-CD 19 binding motif is Ab 11. Table 41 provides the experimental conditions or groups mentioned in the subsequent tables of this example. CAR-T cells were administered at a dose and to multiple mice, as shown in table 41. All CAR-T cells were administered intravenously (QDx 1). All mice also received a 36. mu.g dose of human IL-2((Q12Hx2) QDx 3).

Table 41

A number of indicators were monitored including treatment-related weight change, treatment-related mortality, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased lifespan (% ILS). The results are shown in table 42 below. Anti-tumor efficacy was observed.

Watch 42

Example 12

The present embodiments provide methods for (i) a CAR-T cell comprising a dicistronic CAR comprising an anti-CD 19 CAR and an anti-CD 20 CAR; and (ii) data for anti-tumor efficacy of a bispecific CAR comprising an anti-CD 19 binding motif and an anti-CD 20 binding motif. Bicistronic and bispecific anti-CD 20/anti-CD 19 CAR-T cells were tested against disseminating luciferase-expressing Raji B cell lymphoma tumors in NSG mice. Blood samples were monitored weekly for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. Whole blood was drawn for analysis on days 7, 13, 20, 27, 34 and 40. BLI was performed on days 5, 12, 19, 26, 33, and 40. The study was terminated on day 48.

The CAR-T cells of this study, as shown in table 43 below, were bicistronic or bispecific CAR-T cells comprising an anti-CD 20 binding motif and an anti-CD 19 binding motif (Ab 11). Table 43 provides the experimental conditions or groups mentioned in the subsequent tables of this example. CAR-T cells were administered at a dose of 6.0E +06, with each CAR-T cell type administered to six mice. All CAR-T cells were administered intravenously (QDx 1). All mice also received a 36. mu.g dose of human IL-2 (Q12Hx2) QDx3 intraperitoneally.

Watch 43

1	PBS control
		2	Simulated CAR-T cell control (non-transduced T cells) (60.0E +06 cells)
3	CAR-T cells (2.6E +06 cells) comprising an anti-CD 19 control CAR
		4	CAR-T cells comprising an anti-CD 19 CAR with a linker according to SEQ ID NO 247
5	CAR-T cells comprising an anti-CD 19 CAR with a G4S linker
		6	CAR-T cells comprising a bicistronic CAR comprising binding motif #2
7	CAR-T cells comprising a dicistronic CAR comprising binding motif #9
		8	CAR-T cells comprising a dicistronic CAR comprising binding motif #14
9	CAR-T cells comprising a bispecific CAR comprising binding motif #2
		10	CAR-T cells comprising a bispecific CAR comprising binding motif #9
11	CAR-T cells comprising a bispecific CAR comprising binding motif #14
		12	CAR-T cells comprising control bispecific anti-CD 20/anti-CD 19 CAR
13	CAR-T cells comprising control bispecific anti-CD 20/anti-CD 19 CAR

A number of indicators were monitored including treatment-related weight change, treatment-related mortality, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased lifespan (% ILS). The results are shown in table 44 below, noting that the data reflects monitoring by terminating the experiment on day 48.

All treatments were tolerated. All treatment regimens, excluding non-transduced and LG cells, resulted in a median tumor growth delay of at least 21 days, an Increased Lifespan (ILS) > 223%, a median% T/C of approximately 0% at day 12, and many partial and/or complete tumor regressions resulting therefrom.

Watch 44

Example 13

This example provides data relating to the anti-tumor efficacy of CAR-T cells comprising a monovalent anti-CD 20 CAR. anti-CD 20 CAR-T cells were tested against disseminating luciferase-expressing Raji B cell lymphoma tumor (CD19WT) in NSG mice. Blood samples were monitored weekly for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. BLI was performed on days 5, 12, 21, 28, 33, 40 and 47. The study was terminated on day 55. CAR-T cells of this study were administered at the doses and number of mice shown in table 45, as shown in table 45 below. Table 45 provides the experimental conditions or groups mentioned in the subsequent tables of this example. All CAR-T cells were administered intravenously (QDx 1). All mice received a 36. mu.g dose of human IL-2((Q12Hx2) QDx 3).

TABLE 45

A number of indicators were monitored including treatment-related weight change, treatment-related mortality, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased lifespan (% ILS). The results are shown in table 46 below.

TABLE 46

Example 14

This example provides data relating to the anti-tumor efficacy of CAR-T cells comprising a dicistronic CAR comprising an anti-CD 19 CAR and an anti-CD 20 CAR, and data relating to the anti-tumor efficacy of CAR-T cells comprising a monovalent anti-CD 20 CAR. CAR-T cells were tested against disseminating luciferase-expressing Raji B cell lymphoma tumors in NSG mice. Blood samples were monitored weekly for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. BLI was performed on days 5, 12, 19, 26, 33, and 40.

CAR-T cells of this study are shown in table 47 below. Table 47 provides the experimental conditions or groups mentioned in the subsequent tables of this example. Groups 1-10 contained control and monospecific CAR-T cells only, while groups 11-16 contained bicistronic CAR-T cells. Bicistronic CAR-T cells comprise an anti-CD 20 CAR and an anti-CD 19 CAR (Ab11 CAR) as shown in table 39. Each condition shown in table 47 contained 6 mice. All CAR-T cells were administered intravenously at the indicated dose (QDx 1). All mice also received a 36. mu.g dose of human IL-2 (Q12Hx2) QDx3 intraperitoneally.

Watch 47

A number of indicators were monitored including treatment-related weight change, treatment-related mortality, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased lifespan (% ILS). The results are shown in table 48 below. Mice were studied to be well-tolerated by all CAR-T cell therapies.

Watch 48

Example 15

This example provides data relating to the anti-tumor efficacy of CAR-T cells comprising a dicistronic CAR comprising an anti-CD 19 CAR and an anti-CD 20 CAR, and data relating to the anti-tumor efficacy of CAR-T cells comprising a monovalent anti-CD 20 CAR. CAR-T cells were tested against the luciferase-expressing NALM6 human acute lymphoblastic leukemia model in NSG mice. The blood samples were monitored for CAR-T persistence. Responses were assessed based on bioluminescence imaging (BLI) and survival endpoint. Whole blood was drawn for analysis on days 4, 11, 20, 25, 32 and 39. BLI was performed on days 3, 10, 18, 24, 31, 38, and 45. The study was terminated on day 48.

CAR-T cells of this study are shown in table 49 below. Table 49 provides the experimental conditions or groups mentioned in the subsequent tables of this example. Groups 1-8 contained only control and monospecific CAR-T cells, while groups 9-12 contained bicistronic CAR-T cells. The bicistronic CAR-T cells comprise an anti-CD 20 CAR and an anti-CD 19 CAR (Ab11 CAR) as shown in table 49. Each condition shown in table 49 contained 6 mice. All CAR-T cells were administered intravenously at the indicated dose (QDx 1).

Watch 49

A number of indicators were monitored including treatment-related weight change, treatment-related mortality, median tumor growth delay, median tumor burden (% T/C) compared to control, percentage of animals showing partial tumor regression (% PR), percentage of animals showing complete tumor regression (% CR), percentage of tumor-free survivors (% TFS), and percentage of increased lifespan (% ILS). The results are shown in table 50 below. The study mice were able to tolerate all CAR-T cell treatments.

Watch 50

Example 16

This example provides data relating to sequences used as components of a CAR. Four anti-CD 20 binding motifs were tested in combination with 4 hinges (CD8 hinge (8k), truncated CD28 hinge (28T), truncated CD28 hinge with G4S linker; and IgG4 hinge (I4)). All bispecific and bicistronic CARs contained an Ab11 anti-CD 19 binding motif. CD4 ⁺And CD8⁺T cells were isolated by positive selection from apheresis material from healthy donors and used to generate either an anti-CD 20 monovalent or anti-CD 20/anti-CD 19 bicistronic CAR T cell product. T cells were activated with bound anti-CD 3 and soluble CD28 antibodies and transduced with lentiviral vectors encoding CAR constructs. As a control, non-transduced (NTD) T cells were generated in parallel from the same donor T cells. On the day of harvest (8-10 days of manufacture), CAR T cell products were stained and analyzed by flow cytometry to assess transduction efficiency and for co-culture assays. The transduction efficiency of T cells with a monovalent CAR-encoding vector and a bicistronic CAR-encoding vector is monitored.

To determine the T cell transduction efficiency of vectors encoding monovalent CARs, CAR-T products were stained with a panel of antibodies (anti-CD 3, anti-CD 4, anti-CD 8, and anti-linker) in the presence of a fixable viability dye and analyzed by flow cytometry to assess the percentage of CAR-positive cells that survived. An anti-linker antibody is an antibody that binds to the linker between the heavy and light chains of the anti-CD 20 CAR binding motif and is used to measure transduction efficiency. Controls included non-transduced cells (NTD), cells transduced with retrovirus containing control anti-CD 19 binding agent, and cells transduced with control anti-CD 20 binding agent (Ab12 binding motif).

To determine the T cell transduction efficiency of the bicistronic CAR-encoding vectors, CAR-T products were stained with a panel of antibodies (comprising anti-CD 3, anti-CD 4, anti-CD 8, anti-idiotype, and anti-linker) in the presence of a fixable viability dye and analyzed by flow cytometry to assess the percentage of CAR-positive cells that survived. The anti-idiotype antibody binds to the binding motif of Ab11 anti-CD 19 binding motif. Thus, the anti-idiotype antibody binds to an anti-CD 19 CAR. It was used to measure the transduction efficiency of anti-CD 19 CARs. Anti-linker antibodies were used to measure transduction efficiency of anti-CD 20 CARs. Controls included non-transduced cells (NTD), cells transduced with retroviruses containing control anti-CD 19 binding agents, and cells transduced with control anti-CD 20/anti-CD 19 bispecific CARs (Ab13/Ab14 bispecific; Ab11/Ab12 bispecific).

To facilitate tracking of T cells in culture, CellTrace was used according to the manufacturer's instructions^TMThe Violet (CTV) reagent labels CAR-T cells, which are then washed with R-10% medium. To facilitate tracking of cells expressing the CAR-T target antigen ("target cells"), the target cells are engineered to express luciferase. Luciferase-expressing target cells comprise Nalm6 and Raji, both of which express both CD19 and CD20 antigens. In addition, Nalm6 and Raji cells (knock-out cells, or KO) that do not express CD19 or CD20 were prepared. These CD19KO and CD20KO cells were selected from Nalm6 and Raji parental cell clones and expressed CD20 but not CD19, or CD19 but not CD20, respectively. CD19KO and CD20KO strains were generated and used as controls to functionally evaluate the antigen binding of each CAR of bicistronic anti-CD 20/anti-CD 19 CAR-expressing cells.

T cell mediated cytotoxicity was measured as a function of the decrease in target luciferase signal in co-culture wells compared to the signal emitted by target cells plated alone. D-luciferin was primed on day 4 after the start of co-cultureThe material was added to the co-culture wells at a final concentration of 0.14mg/mL, and the plates were incubated at 37 ℃ for 10 minutes in the dark. In Varioskan^TMLUX orAnd reading the luminous signal in the Flash multi-mode microplate reader. T cell mediated cytotoxicity was calculated as follows: % cytotoxicity ═ luciferase signal of [ 1- (sample of interest/target-alone control ]]*100。

Cytokine production was measured after 16 hours of co-culture at which time the supernatant was collected and analyzed for cytokine levels using the Meso Scale Discovery V-PLEX pro-inflammatory group 1 human kit according to the manufacturer's instructions. Supernatants from co-cultures of T cell products plated at a 1:1E: T ratio with antigen expressing target cells were analyzed for levels of interferon gamma (IFN-. gamma.), IL-2, tumor necrosis factor alpha (TNF-. alpha.), and IL-10 secretion mediated by antigen conjugation. All samples were diluted to within the detection range. The level of each cytokine is reported in pg/mL, and the lower and upper limit of quantitation for each assay is reported.

To determine T cell activation after 16 hours of co-culture, T cell products plated with antigen-positive target cells at a specific E: T ratio were harvested, stained with a panel of antibody fluorophores to identify T cells (CD3, CD4, CD8) and 4-1BB (an activation marker), and analyzed by flow cytometry. The fixable viability dye allows analysis of surviving cells. Events were in live cells (vital dye negative), lymphocytes (using forward scatter [ FSC ]]Regio-and-sidescatter [ SSC]Region map), single cells (using FSC region and FSC height map), then systematically gated on T cells (CD3 +). The level of activation of T cells (i.e., 4-1 BB) is then analyzed⁺Percentage of cells); the 4-1BB gating threshold was set based on the level of NTD control T cell expression.

The proliferative capacity of the T cell product was determined by flow cytometry analysis of cell division-driven dilution of CTV dye in response to antigen-expressing target cells, compared to NTD control T cells. On day 4 after the start of co-culture, T cell products plated at a 3:1E: T ratio with target cells expressing antigen were harvested, stained with a panel of antibody fluorophores (CD3, CD4, CD8) in the presence of a fixable viability dye to identify viable T cells and analyzed by flow cytometry. The percentage of proliferating cells and the Mean Fluorescence Intensity (MFI) of the CTV signal are reported. The decrease in MFI of CTV is proportional to the number of rounds of cell division experienced by the product.

The CAR-T cells used in this example comprise the CARs identified in table 51 below. Table 51 provides the experimental conditions or groups mentioned in the subsequent tables of this example. Transduction efficiencies are shown in table 52. Day 4 cytotoxicity in Nalm6(CD19 +; CD20 low), Nalm6CD19KO (CD 19-; CD20+), Raji (CD19+, CD20+) and Raji CD19KO (CD19-, CD20+) is shown in tables 53A-53D. Cytokine production 14 hours after co-culture at 1:3E: T ratio in Nalm6CD19KO target cells is shown in tables 54A-54D, measured in pg/mL (2 replicates/condition). T cell activation as measured by upregulation of 41BB (percentage of 41BB + live T cells) 16 hours after co-culture is shown in tables 55A-D (2 replicates per condition). Proliferation of T cells at day 4 after co-culture is shown in table 56 (percentage of proliferating CD3+, 2 replicates per condition).

Watch 51

Table 52

Group of	1	2	5	6	7	8
							% CD19 CAR + live T cells	0.56	59.52	2.35	47.54	29.05	56.77
% CD20 CAR + live T cells	0.09	69.3	75.76	49.33	40.13	60.99
							Group of	9	10	11	12	13	14
% CD19 CAR + live T cells	52.72	50.2	44.72	40.28	38.99	34.12
							% CD20 CAR + live T cells	46.42	45.57	41.36	37.04	37.15	34.7
Group of	15	16	17	18	19	20
							% CD19 CAR + live T cells	34.87	51.32	28.89	41.69	41.34	36.22
% CD20 CAR + live T cells	33.78	47.97	28.87	41.82	41.95	35.14
							Group of	21	22	23	24
% CD19 CAR + live T cells	37.07	30.62	32.18	39.93
							% CD20 CAR + live T cells	38.69	41	41.23	46.43

TABLE 53A

TABLE 53B

Table 53C

Table 53D

TABLE 54A

Table 54B

Table 54C

Table 54D

TABLE 55A

TABLE 55B

Table 55C

TABLE 55D

Watch 56

Example 17

The comparison of the hinges was performed in an anti-CD 20 monovalent CAR. The data provided in this example were obtained using the methods described in the examples above. The CAR-T cells used in this example comprise the CARs shown in table 57 below. Table 57 provides the experimental conditions or groups mentioned in the subsequent tables of this example. This example utilizes four anti-CD 20 binding motifs, shown in this example as binding motif a, binding motif B, binding motif C, and binding motif D. The hinges tested included a truncated CD28 hinge (28T), a CD8 hinge (8k), and an IgG4 hinge (I4).

Transduction efficiencies are shown in table 58. Day 4 cytotoxicity in Raji (CD19+, CD20 high), Raji CD20KO (CD19+, CD20-), Namalwa (CD19+, CD20 low), and Nalm6(CD19+, CD20 low) are shown in tables 59 and 60. Cytokine production 14 hours after co-culture at 1:1E: T ratio is shown in tables 61A-D. T cell activation measured by upregulation of 41BB 16 hours after co-cultivation in the case of E: T1: 1 or 1:4 is shown in tables 62 and 63, respectively. T cell proliferation 4 days after co-culture in E: T1: 1 or 1:4 cases is shown in tables 64 and 65, respectively.

Watch 57

Watch 58

Group of	Transduction efficiency (% CAR +)
		1	0.15
2	62.3
		3	56.3
4	89.2
		5	51.6
6	66.5
		7	71.6
8	73.9
		9	77.0
10	85.4
		11	66.8
12	80.8
		13	84.8

Watch 59

Watch 60

TABLE 61A

Table 61B

Table 61C

Table 61D

Watch 62

Table 63

Table 64

Table 65

Example 18

Table 66: exemplary binding motif sequences

Example 19

Table 67: exemplary hinge sequence

Example 20

Table 68: exemplary nucleotide sequences encoding binding motifs, hinges, and 41BB costimulatory domains

Example 21

Table 69: exemplary anti-CD 20/anti-CD 19 dicistronic CAR nucleotide and amino acid sequences shown in SEQ ID NOS: 291 and 292.

Example 22

Table 70: exemplary anti-CD 20/anti-CD 19 bispecific CAR and components thereof

Example 23

TABLE 71 exemplary Joint

While various embodiments have been described, it is apparent that the disclosure and examples may provide other embodiments that utilize or are encompassed by the compositions and methods described herein. It is therefore to be understood that the scope of the invention is defined by what can be understood from the disclosure and the appended claims, and not by the embodiments shown by way of example.

Sequence listing

<110> Kaide pharmaceutical Co., Ltd

ADIMAB, LLC

<120> chimeric antigens and T cell receptors and methods of use

<130> K-1074

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<150> 62/778,893

<151> 2018-12-12

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<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 6

Ile Asp His Ser Gly Ser Thr

1 5

<210> 7

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 7

Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 8

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 8

Asp His Ser Gly Ser

1 5

<210> 9

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 9

Ala Arg Gly Gly Gly Ser Trp Tyr Ser Asn Trp Phe Asp Pro

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 10

Gly Gly Gly Ser Trp Tyr Ser Asn Trp Phe Asp Pro

1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 11

Gly Gly Gly Ser Trp Tyr Ser Asn Trp Phe Asp Pro

1 5 10

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Arg Ser Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 13

gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccagcag gaccgaagtc tccctcctac ttttggcgga 300

gggaccaagg ttgagatcaa a 321

<210> 14

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 14

Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala

1 5 10

<210> 15

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 15

Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala

1 5 10

<210> 16

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 16

Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala

1 5 10

<210> 17

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 17

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 18

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 19

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 20

Gln Gln Asp Arg Ser Leu Pro Pro Thr

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 21

Gln Gln Asp Arg Ser Leu Pro Pro Thr

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 22

Gln Gln Asp Arg Ser Leu Pro Pro Thr

1 5

<210> 23

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 23

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Ile

20 25 30

His Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asp Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Leu Gly Gln Glu Ser Ala Thr Tyr Leu Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 24

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggtatccact ggaactggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggac atcgacacaa gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatcc gtagacacgt ccaagaacca gttctccctg 240

aagctgagtt ctgtgaccgc cgcagacacg gcggtgtact actgcgccag attgggacag 300

gagtcagcca cctatctcgg aatggacgta tggggccagg gaacaactgt caccgtctcc 360

tca 363

<210> 25

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 25

Gly Gly Ser Phe Ser Gly Ile His

1 5

<210> 26

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 26

Gly Ile His Trp Asn

1 5

<210> 27

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 27

Gly Gly Ser Phe Ser Gly

1 5

<210> 28

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 28

Ile Asp Thr Ser Gly Ser Thr

1 5

<210> 29

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 29

Asp Ile Asp Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 30

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 30

Asp Thr Ser Gly Ser

1 5

<210> 31

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 31

Ala Arg Leu Gly Gln Glu Ser Ala Thr Tyr Leu Gly Met Asp Val

1 5 10 15

<210> 32

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 32

Leu Gly Gln Glu Ser Ala Thr Tyr Leu Gly Met Asp Val

1 5 10

<210> 33

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 33

Leu Gly Gln Glu Ser Ala Thr Tyr Leu Gly Met Asp Val

1 5 10

<210> 34

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 34

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Leu Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 35

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 35

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcagct ctacacctac 300

cctttcactt ttggcggagg gaccaaggtt gagatcaaa 339

<210> 36

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 36

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 37

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 37

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 38

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 38

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 39

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 40

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 40

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 41

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 41

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 42

Gln Gln Leu Tyr Thr Tyr Pro Phe Thr

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 43

Gln Gln Leu Tyr Thr Tyr Pro Phe Thr

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 44

Gln Gln Leu Tyr Thr Tyr Pro Phe Thr

1 5

<210> 45

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 45

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 46

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 46

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtagtagtt actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240

tccctgaagc tgagttctgt gaccgccgca gacacggcgg tgtactactg cgccagagag 300

actgactaca gcagcggaat gggatacgga atggacgtat ggggccaggg aacaactgtc 360

accgtctcct ca 372

<210> 47

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 47

Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr

1 5 10

<210> 48

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 48

Ser Ser Ser Tyr Tyr Trp Gly

1 5

<210> 49

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 49

Gly Gly Ser Ile Ser Ser Ser Ser

1 5

<210> 50

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 50

Ile Tyr Tyr Ser Gly Ser Thr

1 5

<210> 51

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 51

Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 52

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 52

Tyr Tyr Ser Gly Ser

1 5

<210> 53

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 53

Ala Arg Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val

1 5 10 15

<210> 54

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 54

Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val

1 5 10

<210> 55

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 55

Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val

1 5 10

<210> 56

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 56

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Ala Asp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 57

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattaac agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgccagcaa agcctcgccg accctttcac ttttggcgga 300

gggaccaagg ttgagatcaa a 321

<210> 58

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 58

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 59

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 59

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 60

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 60

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 61

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 61

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 62

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 62

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 63

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 63

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 64

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 64

Gln Gln Ser Leu Ala Asp Pro Phe Thr

1 5

<210> 65

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 65

Gln Gln Ser Leu Ala Asp Pro Phe Thr

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 66

Gln Gln Ser Leu Ala Asp Pro Phe Thr

1 5

<210> 67

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 67

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Glu Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Ser Gly His Thr Tyr Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro His Tyr Asp Asp Trp Ser Gly Phe Ile Ile Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 68

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 68

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttaaa gaatatggta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgctt acagtggtca cacatactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240

atggagctga ggagcctgag atctgacgac acggcggtgt actactgcgc cagagggcct 300

cactacgacg actggagcgg atttatcata tggttcgacc catggggaca gggtacattg 360

gtcaccgtct cctca 375

<210> 69

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 69

Gly Tyr Thr Phe Lys Glu Tyr Gly

1 5

<210> 70

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 70

Glu Tyr Gly Ile Ser

1 5

<210> 71

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 71

Gly Tyr Thr Phe Lys Glu

1 5

<210> 72

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 72

Ile Ser Ala Tyr Ser Gly His Thr

1 5

<210> 73

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 73

Trp Ile Ser Ala Tyr Ser Gly His Thr Tyr Tyr Ala Gln Lys Leu Gln

1 5 10 15

<210> 74

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 74

Ser Ala Tyr Ser Gly

1 5

<210> 75

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 75

Ala Arg Gly Pro His Tyr Asp Asp Trp Ser Gly Phe Ile Ile Trp Phe

1 5 10 15

Asp Pro

<210> 76

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 76

Gly Pro His Tyr Asp Asp Trp Ser Gly Phe Ile Ile Trp Phe Asp Pro

1 5 10 15

<210> 77

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 77

Gly Pro His Tyr Asp Asp Trp Ser Gly Phe Ile Ile Trp Phe Asp Pro

1 5 10 15

<210> 78

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 78

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 79

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 79

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccttca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacaggt ttcctcctac ctttggccaa 300

gggaccaagg ttgagatcaa a 321

<210> 80

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 80

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 81

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 81

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 82

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 82

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 83

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 83

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 84

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 84

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 85

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 85

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 86

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 86

Gln Gln Ser Tyr Arg Phe Pro Pro Thr

1 5

<210> 87

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 87

Gln Gln Ser Tyr Arg Phe Pro Pro Thr

1 5

<210> 88

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 88

Gln Gln Ser Tyr Arg Phe Pro Pro Thr

1 5

<210> 89

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 89

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Pro

20 25 30

Asp His Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Ala Ser Gly Ser Thr Phe Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 90

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 90

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtcccgacc actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggtccatct acgccagtgg gagcaccttc 180

tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgccgcg gacacggcgg tgtactactg cgccagagag 300

actgactaca gcagcggaat gggatacgga atggacgtat ggggccaggg aacaactgtc 360

accgtctcct ca 372

<210> 91

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 91

Gly Gly Ser Ile Ser Ser Pro Asp His Tyr

1 5 10

<210> 92

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 92

Ser Pro Asp His Tyr Trp Gly

1 5

<210> 93

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 93

Gly Gly Ser Ile Ser Ser Pro Asp

1 5

<210> 94

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 94

Ile Tyr Ala Ser Gly Ser Thr

1 5

<210> 95

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 95

Ser Ile Tyr Ala Ser Gly Ser Thr Phe Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 96

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 96

Tyr Ala Ser Gly Ser

1 5

<210> 97

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 97

Ala Arg Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val

1 5 10 15

<210> 98

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 98

Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val

1 5 10

<210> 99

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 99

Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val

1 5 10

<210> 100

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 100

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Ala Asp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 101

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 101

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattaac agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgccagcaa agcctcgccg accctttcac ttttggcgga 300

gggaccaagg ttgagatcaa a 321

<210> 102

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 102

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 103

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 103

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 104

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 104

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 105

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 105

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 106

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 106

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 107

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 107

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 108

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 108

Gln Gln Ser Leu Ala Asp Pro Phe Thr

1 5

<210> 109

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 109

Gln Gln Ser Leu Ala Asp Pro Phe Thr

1 5

<210> 110

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 110

Gln Gln Ser Leu Ala Asp Pro Phe Thr

1 5

<210> 111

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 111

Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Asp Thr Glu

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Tyr Phe Asn Asp Gln Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Thr Gly Tyr Ser Arg Trp Tyr Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 112

<211> 369

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 112

cagatcacct tgaaggagtc tggtcctacg ctggtgaaac ccacacagac cctcacgctg 60

acctgcacct tctctgggtt ctcactcgac actgaaggag tgggtgtggg ctggatccgt 120

cagcccccag gaaaggccct ggagtggctt gcactcattt atttcaatga tcaaaagcgc 180

tacagcccat ctctgaagag caggctcacc atcaccaagg acacctccaa aaaccaggtg 240

gtccttacaa tgaccaacat ggaccctgtg gacacggcgg tgtactactg cgccagagac 300

acgggataca gccgatggta ctacggcatg gatgtatggg gccagggaac aactgtcacc 360

gtctcctca 369

<210> 113

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 113

Gly Phe Ser Leu Asp Thr Glu Gly Val Gly

1 5 10

<210> 114

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 114

Thr Glu Gly Val Gly Val Gly

1 5

<210> 115

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 115

Gly Phe Ser Leu Asp Thr Glu Gly

1 5

<210> 116

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 116

Ile Tyr Phe Asn Asp Gln Lys

1 5

<210> 117

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 117

Leu Ile Tyr Phe Asn Asp Gln Lys Arg Tyr Ser Pro Ser Leu Lys Ser

1 5 10 15

<210> 118

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 118

Tyr Phe Asn Asp Gln

1 5

<210> 119

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 119

Ala Arg Asp Thr Gly Tyr Ser Arg Trp Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 120

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 120

Asp Thr Gly Tyr Ser Arg Trp Tyr Tyr Gly Met Asp Val

1 5 10

<210> 121

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 121

Asp Thr Gly Tyr Ser Arg Trp Tyr Tyr Gly Met Asp Val

1 5 10

<210> 122

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 122

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ala Tyr Pro Ile

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 123

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 123

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcagcag gcatacgcct accctatcac ttttggcgga 300

gggaccaagg ttgagatcaa a 321

<210> 124

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 124

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 125

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 125

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 126

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 126

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 127

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 127

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 128

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 128

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 129

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 129

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 130

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 130

Gln Gln Ala Tyr Ala Tyr Pro Ile Thr

1 5

<210> 131

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 131

Gln Gln Ala Tyr Ala Tyr Pro Ile Thr

1 5

<210> 132

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 132

Gln Gln Ala Tyr Ala Tyr Pro Ile Thr

1 5

<210> 133

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 133

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Glu Lys Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr His Ser Gly Leu Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 134

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 134

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcgaa aaatactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atctaccata gtggactcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gcggtgtact actgcgccag ggtcagatac 300

gacagcagcg actcctacta ctatagctac gattatggaa tggacgtatg gggccaggga 360

acaactgtca ccgtctcctc a 381

<210> 135

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 135

Gly Gly Ser Phe Glu Lys Tyr Tyr

1 5

<210> 136

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 136

Lys Tyr Tyr Trp Ser

1 5

<210> 137

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 137

Gly Gly Ser Phe Glu Lys

1 5

<210> 138

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 138

Ile Tyr His Ser Gly Leu Thr

1 5

<210> 139

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 139

Glu Ile Tyr His Ser Gly Leu Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 140

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 140

Tyr His Ser Gly Leu

1 5

<210> 141

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 141

Ala Arg Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp

1 5 10 15

Tyr Gly Met Asp Val

20

<210> 142

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 142

Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr Gly

1 5 10 15

Met Asp Val

<210> 143

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 143

Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr Gly

1 5 10 15

Met Asp Val

<210> 144

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 144

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 145

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 145

gacatcgtgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctagccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcagtc ctactccttc 300

ccttggactt ttggcggagg gaccaaggtt gagatcaaa 339

<210> 146

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 146

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 147

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 147

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 148

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 148

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 149

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 149

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 150

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 150

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 151

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 151

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 152

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 152

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 153

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 153

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 154

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 154

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 155

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 155

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Arg Tyr

20 25 30

Val Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Ser Ser Gly Lys Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 156

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 156

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt cgatacgtat ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcgactcca gtggaaaaac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gcggtgtact actgcgccag ggtcagatac 300

gacagcagcg actcctacta ctatagctac gattatggaa tggacgtatg gggccaggga 360

acaactgtca ccgtctcctc a 381

<210> 157

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 157

Gly Gly Ser Phe Ser Arg Tyr Val

1 5

<210> 158

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 158

Arg Tyr Val Trp Ser

1 5

<210> 159

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 159

Gly Gly Ser Phe Ser Arg

1 5

<210> 160

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 160

Ile Asp Ser Ser Gly Lys Thr

1 5

<210> 161

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 161

Glu Ile Asp Ser Ser Gly Lys Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 162

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 162

Asp Ser Ser Gly Lys

1 5

<210> 163

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 163

Ala Arg Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp

1 5 10 15

Tyr Gly Met Asp Val

20

<210> 164

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 164

Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr Gly

1 5 10 15

Met Asp Val

<210> 165

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 165

Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr Gly

1 5 10 15

Met Asp Val

<210> 166

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 166

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 167

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 167

gacatcgtgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctagccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcagtc ctactccttc 300

ccttggactt ttggcggagg gaccaaggtt gagatcaaa 339

<210> 168

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 168

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 169

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 169

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 170

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 170

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 171

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 171

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 172

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 172

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 173

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 173

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 174

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 174

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 175

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 175

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 176

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 176

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 177

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 177

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Arg Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 178

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 178

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttctcc ggttacgcat ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcgaccatc gaggattcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gcggtgtact actgcgccag ggtcagatac 300

gacagcagcg actcctacta ctatagctac gattatggaa tggacgtatg gggccaggga 360

acaactgtca ccgtctcctc a 381

<210> 179

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 179

Gly Gly Ser Phe Ser Gly Tyr Ala

1 5

<210> 180

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 180

Gly Tyr Ala Trp Ser

1 5

<210> 181

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 181

Gly Gly Ser Phe Ser Gly

1 5

<210> 182

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 182

Ile Asp His Arg Gly Phe Thr

1 5

<210> 183

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 183

Glu Ile Asp His Arg Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 184

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 184

Asp His Arg Gly Phe

1 5

<210> 185

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 185

Ala Arg Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp

1 5 10 15

Tyr Gly Met Asp Val

20

<210> 186

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 186

Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr Gly

1 5 10 15

Met Asp Val

<210> 187

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 187

Val Arg Tyr Asp Ser Ser Asp Ser Tyr Tyr Tyr Ser Tyr Asp Tyr Gly

1 5 10 15

Met Asp Val

<210> 188

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 188

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 189

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 189

gacatcgtgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctagccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcagtc ctactccttc 300

ccttggactt ttggcggagg gaccaaggtt gagatcaaa 339

<210> 190

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 190

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 191

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 191

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 192

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 192

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 193

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 193

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 194

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 194

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 195

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 195

Trp Ala Ser Ser Arg Glu Ser

1 5

<210> 196

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 196

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 197

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 197

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 198

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 198

Gln Gln Ser Tyr Ser Phe Pro Trp Thr

1 5

<210> 199

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 199

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Gln Lys Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Thr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Gly Arg Tyr Ser Tyr Gly Tyr Tyr Ile Thr Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 200

<211> 369

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 200

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttccaa aaatactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcgacacca gtggattcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gcggtgtact actgcgccag agtgggaagg 300

tacagctacg gatactatat caccgcattc gacatatggg gtcagggtac aactgtcacc 360

gtctcctca 369

<210> 201

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 201

Gly Gly Ser Phe Gln Lys Tyr Tyr

1 5

<210> 202

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 202

Lys Tyr Tyr Trp Ser

1 5

<210> 203

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 203

Gly Gly Ser Phe Gln Lys

1 5

<210> 204

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 204

Ile Asp Thr Ser Gly Phe Thr

1 5

<210> 205

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 205

Glu Ile Asp Thr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 206

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 206

Asp Thr Ser Gly Phe

1 5

<210> 207

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 207

Ala Arg Val Gly Arg Tyr Ser Tyr Gly Tyr Tyr Ile Thr Ala Phe Asp

1 5 10 15

Ile

<210> 208

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 208

Val Gly Arg Tyr Ser Tyr Gly Tyr Tyr Ile Thr Ala Phe Asp Ile

1 5 10 15

<210> 209

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 209

Val Gly Arg Tyr Ser Tyr Gly Tyr Tyr Ile Thr Ala Phe Asp Ile

1 5 10 15

<210> 210

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 210

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Ser Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 211

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 211

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcagca ctactccttc 300

cctttcactt ttggcggagg gaccaaggtt gagatcaaa 339

<210> 212

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 212

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 213

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 213

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 214

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 214

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 215

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 215

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 216

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 216

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 217

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 217

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 218

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 218

Gln Gln His Tyr Ser Phe Pro Phe Thr

1 5

<210> 219

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 219

Gln Gln His Tyr Ser Phe Pro Phe Thr

1 5

<210> 220

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 220

Gln Gln His Tyr Ser Phe Pro Phe Thr

1 5

<210> 221

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 221

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 222

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 222

gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60

acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120

ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180

tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300

tacggtggta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360

<210> 223

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 223

Gly Val Ser Leu Pro Asp Tyr Gly

1 5

<210> 224

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 224

Asp Tyr Gly Val Ser

1 5

<210> 225

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 225

Gly Val Ser Leu Pro Asp Tyr

1 5

<210> 226

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 226

Ile Trp Gly Ser Glu Thr Thr

1 5

<210> 227

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 227

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 228

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 228

Trp Gly Ser Glu Thr

1 5

<210> 229

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 229

Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 230

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 230

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 231

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 231

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 232

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 232

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 233

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 233

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggactaagt tggaaataac a 321

<210> 234

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 234

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 235

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 235

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 236

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 236

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 237

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 237

His Thr Ser Arg Leu His Ser

1 5

<210> 238

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 238

His Thr Ser Arg Leu His Ser

1 5

<210> 239

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 239

His Thr Ser Arg Leu His Ser

1 5

<210> 240

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 240

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 241

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 241

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 242

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 242

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 243

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 243

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 244

<211> 735

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 244

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag 360

ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 420

cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 480

tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 540

accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 600

agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 660

gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 720

gtcaccgtct cctca 735

<210> 245

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 245

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 246

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Oligonucleotides

<400> 246

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210> 247

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 247

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 248

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Oligonucleotides

<400> 248

ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54

<210> 249

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 249

Met Glu Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly

1 5 10 15

Val His Ser

<210> 250

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 250

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 251

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 251

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg ctattactgg 180

agctggatcc ggcagcctcc tggaaaagga ttagaatgga tcggcgagat agaccacagc 240

gggagcacaa actacaaccc cagcctgaaa tcgcgggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag gcggaggctc ctggtacagc aactggttcg atccttgggg ccaaggcacc 420

atggtgaccg tttccagcgg ctctacaagc ggcagcggga aacctggttc tggagagggc 480

agcacaaagg gcgacatcca gatgacacag agccccagca cccttagcgc ctctgtggga 540

gatagggtta ccattacctg cagggcttcc cagagcatca gcagctggct ggcatggtat 600

caacagaagc ctggcaaggc tcccaagctg ctcatctatg acgcctccag cctggaaagc 660

ggggttccct ccagatttag cggctcaggc tccggaacag agttcaccct taccatctct 720

agcctgcaac ccgacgactt cgctacttat tactgtcaac aagacagaag cttgcccccc 780

acattcggcg gagggaccaa ggttgagatc aag 813

<210> 252

<211> 834

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 252

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg catccactgg 180

aactggatcc ggcagcctcc tggcaaaggc cttgaatgga tcggcgatat cgacaccagc 240

ggctccacca actacaaccc cagcctgaaa tcgagggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagac tgggccagga aagcgctacc taccttggca tggatgtgtg ggggcagggc 420

accaccgtta ctgttagctc tggctcaaca agcggcagcg gcaagcctgg ctcaggagaa 480

ggaagcacaa agggcgacat tgtaatgact cagagccccg acagcctggc cgttagctta 540

ggcgaaaggg ctacaatcaa ttgcaagagc agccagagcg ttctgtacag cagcaacaac 600

aagaactacc tcgcatggta tcaacagaag ccaggccagc ctcccaagct gctcatctac 660

tgggcttcca ccagagagag cggggttccc gatagattct ccggctccgg ttctggaaca 720

gatttcacgc tcacaatcag cagcttacag gccgaggatg tggctgtcta ctattgtcag 780

cagttgtaca cctacccctt cacattcggc ggaggcacca aggttgagat caag 834

<210> 253

<211> 825

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 253

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca gcttcagctc caagagagcg gacctggctt agtgaagccc 120

agcgaaaccc tgtccctcac ctgcaccgtt tctggcggaa gcatcagcag ctccagctat 180

tactggggat ggatcaggca gccccctggc aagggtttag aatggatcgg ctcgatatat 240

tactccggca gcacctacta taaccccagc ttgaagagcc gggttaccat ttctgtggac 300

acatcaaaga accagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcggcatgg gctacggcat ggatgtgtgg 420

ggacaaggaa ccaccgttac tgtgagcagc ggttccacca gcggctcagg caagcctggc 480

tcaggagaag gaagcaccaa gggggatata cagatgacac agagcccctc cagcctgtcc 540

gccagcgttg gcgatcgtgt aacgatcacc tgccgggcct ctcagagcat caactcctac 600

ctcaattggt atcaacagaa gccaggcaag gcccccaaat tactcatcta cgccgccagc 660

agcttacaga gcggggttcc ctctagattc tccggctccg gttctggaac agatttcacc 720

ctcactatct ccagcttgca gcccgaggat ttcgccactt attactgtca gcagagcctg 780

gccgacccct tcacattcgg cggaggcaca aaggttgaga tcaag 825

<210> 254

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 254

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagctt gtgcagagcg gagctgaagt taagaagcct 120

ggcgcctctg tgaaggttag ctgcaaggcc agcggctaca cattcaagga atatggcatc 180

tcctgggtta ggcaggctcc cggccaaggc ttagaatgga tgggctggat ctccgcctac 240

tccggccaca cctactacgc ccagaagctt cagggcaggg ttaccatgac caccgacacc 300

agcacctcta ccgcctatat ggagctgagg agcctgagat cggacgacac agctgtgtat 360

tactgcgcca gaggccccca ctacgacgac tggtctggat ttatcatctg gttcgacccc 420

tgggggcagg gcaccctggt cacagtttct tctggctcca ccagcggaag cggcaagcca 480

ggctcaggcg aaggatctac aaaaggcgac atccaaatga cacagagccc cagcagcttg 540

agcgcctccg ttggcgacag agttacaatc acctgcaggg cctctcagag catcagcagc 600

tatttgaatt ggtatcaaca gaagccagga aaggccccta agctgctcat ctacgctgcc 660

agctcgctcc aatctggcgt tcctagcaga tttagcggct ccggcagcgg cacagacttt 720

actcttacca ttagctccct gcagcccgag gacttcgcta cctactattg ccagcaaagc 780

tacagattcc ctcccacctt tggccagggc acaaaggttg agatcaag 828

<210> 255

<211> 825

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 255

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caagagagcg gacctggctt agtgaagccc 120

agcgaaaccc tgtccctcac ctgcaccgtt tctggcggaa gcatcagctc tcccgaccat 180

tactggggat ggatcaggca gccccctggc aagggtttgg aatggatcgg cagcatctac 240

gccagcggca gcacattcta caacccctcg ctcaaaagca gggttactat ttctgtggac 300

acaagcaaaa atcagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcgggatgg gctacggcat ggatgtgtgg 420

ggacaaggaa ccaccgttac tgtgagcagc ggctccacaa gcggctcagg caagcctggc 480

tcaggagaag gaagcaccaa gggggacatt caaatgaccc aaagcccctc cagcctgtcc 540

gccagcgttg gcgatagggt taccattacc tgcagggcca gccaaagcat caactcctac 600

ctaaattggt atcaacagaa gccaggcaag gcccccaaac tactcattta cgccgccagc 660

agcttacaga gcggggttcc ctctagattc tccggcagcg gttctggaac agatttcact 720

ctcacaatat cttcgctgca gcccgaggat ttcgctacct actattgcca gcaatccctg 780

gccgacccct tcacattcgg cggaggcaca aaggttgaga tcaag 825

<210> 256

<211> 822

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 256

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca gatcacatta aaagagagcg gacctacact ggtgaagccc 120

acccaaacgc ttaccctcac ctgcaccttt agcgggttca gcctggacac agagggcgtt 180

ggcgttggat ggatcaggca gcctcctggc aaagccctcg aatggcttgc cctcatctac 240

ttcaacgacc agaagagata cagcccctcc ttaaaatctc ggctcacaat caccaaagac 300

acaagcaaaa atcaggttgt gctcaccatg accaacatgg accctgtgga caccgctgtg 360

tactactgtg ccagagacac cggctacagc agatggtact acgggatgga cgtttggggc 420

caaggcacca ctgtgaccgt ttccagcggc tctacaagcg gcagcgggaa acctggttct 480

ggagagggca gcacaaaggg cgacatccag atgacgcaat cccccagctc tgtgagcgcc 540

tctgtgggag acagagttac aatcacatgc cgggcctccc agggcatcag ctcttggctg 600

gcatggtatc aacagaagcc tggcaaggct cccaagctgc tcatctatgc cgcctcctcc 660

ttacaatctg gagttccctc caggttcagc gggagcggct caggaacaga cttcaccctt 720

accatctcta gcctgcaacc cgaggacttc gctacttatt actgtcagca ggcctacgcc 780

taccccatca cattcggcgg aggaacaaag gttgagatca ag 822

<210> 257

<211> 852

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 257

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcgagaa atactactgg 180

agctggatcc ggcagcctcc cggcaaaggc ttagaatgga tcggcgagat ttatcacagc 240

gggctcacca actacaaccc cagcctgaaa tctcgagtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttact gtctcctctg gatctaccag cggcagcggc 480

aagcctggat ctggcgaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggctg tgtctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcttggtatc aacagaagcc tggccagccc 660

cctaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tcggtttagc 720

ggctccggct caggaaccga tttcaccctc actatctcca gcctccaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca ag 852

<210> 258

<211> 852

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 258

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagccg ctatgtgtgg 180

agctggatcc ggcagcctcc tggcaaaggc cttgaatgga tcggagagat agacagcagc 240

ggcaagacca actacaaccc cagcctgaaa tcacgcgtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagctccgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttaca gttagctctg gaagcaccag cggctccggc 480

aagcctggat ctggtgaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggctg tgtctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcatggtatc aacagaagcc tggccagcct 660

cccaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tcgctttagc 720

ggcagcggtt ctggcaccga tttcactctt acaatcagca gcttacaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca ag 852

<210> 259

<211> 852

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 259

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg ctacgcttgg 180

agctggatta gacagcctcc tggcaaagga ctagaatgga tcggagagat cgaccacaga 240

ggcttcacca actacaaccc cagcctgaaa tccagagtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccaggg ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttacg gttagctctg gatctaccag cggcagcggc 480

aagcctggct caggagaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggccg tttctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcatggtatc aacagaagcc aggccagcct 660

cccaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tagattttcg 720

ggatcaggct ccggcaccga tttcactctt acgatcagca gcttacaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca ag 852

<210> 260

<211> 840

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 260

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttccagaa atactactgg 180

agctggatcc ggcagcctcc cggcaaaggc ttagaatgga tcggagagat agacaccagc 240

ggcttcacca actacaaccc cagcctgaaa tctagggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttggcagata cagctacggc tactacatca ccgccttcga catttggggc 420

caaggcacca ctgtgaccgt ttccagcgga agcactagcg gcagcgggaa acctggttct 480

ggagagggct caaccaaggg cgacatcgtg atgacacaga gccccgactc tctggctgtg 540

tccctgggag agagagccac catcaactgc aagagcagcc agagcgttct gtacagcagc 600

aacaacaaga actacctggc atggtatcaa cagaagcctg gccagccccc taagctgctc 660

atctactggg cttccaccag agaatcaggc gttccagaca ggttctccgg ctcgggttca 720

ggcacagact tcacccttac catctcttcc ctgcaggccg aagatgtggc cgtttactac 780

tgtcagcagc actacagctt ccctttcaca ttcggcggag gcaccaaggt tgagatcaag 840

<210> 261

<211> 258

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 261

gcagctgctt tcgtgcctgt gttcctgcct gctaagccca ccaccactcc tgctccaaga 60

cctcctaccc ccgctcctac aatcgccagc caacctctga gcctgagacc ggaggcatgc 120

agacctgcgg cagggggagc agttcacaca agaggcttgg acttcgcttg cgacatctac 180

atctgggccc ctctggccgg cacatgcgga gttcttcttc ttagcctggt gatcaccctg 240

tactgcaacc acagaaac 258

<210> 262

<211> 180

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 262

gctgctgcat tggataatga aaaatcgaac ggcacaatca ttcatgtgaa gggcaaacac 60

ctgtgtccca gccccttgtt cccaggacct agcaagcctt tttgggttct cgtggtggtg 120

ggcggcgttc tggcttgcta ctctctactt gtaactgtcg catttattat attctgggtt 180

<210> 263

<211> 186

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 263

ggaggaggag gatctctgga taacgagaaa agcaacggga ccatcattca tgtgaaggga 60

aaacatctgt gtcccagccc cttgttcccc ggacctagca agccgttttg ggttctcgtg 120

gtggtgggcg gcgttctggc ttgctactct ctgcttgtga ccgttgcctt cattatcttc 180

tgggtt 186

<210> 264

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 264

ggaggaggag gatctggtgg aggaggttct ctggacaatg agaaatcaaa tggaacgatc 60

atccatgtga aggggaagca cctctgcccc tctcccctgt ttcctggtcc tagcaagccc 120

ttctgggttt tggtggtcgt gggcggcgtt ctggcttgct acagcctgtt agtgaccgtt 180

gcatttatca tattttgggt t 201

<210> 265

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 265

ggaggaggag gatctggtgg aggaggttct ctggacaatg agaaatcgaa tgggacaatc 60

atccatgtga aggggaagca cctgagcccc tctcccctgt ttcctggtcc tagcaagccc 120

ttctgggttt tggtggtcgt gggcggcgtt ctggccgttt acagcctgtt agtgaccgtt 180

gcttttatca tattttgggt t 201

<210> 266

<211> 201

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 266

ggaggaggag gatctggtgg aggaggttct ctggacaatg aaaagagcaa tggcacaatc 60

atccatgtga aggggaagca cctgaacggc tccgccctgt ttcctggtcc tagcaagcca 120

ttttgggttc tcgtggtggt gggcggcgtt ctggccgttt acagcctgtt agtgaccgtt 180

gcgttcataa tcttctgggt t 201

<210> 267

<211> 216

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 267

ggaggaggag gatctggtgg aggaggttct ggaggaggcg gctctctcga caacgaaaag 60

agcaatggca ccattattca cgttaaaggc aagcatctgt gcccctcccc cctgttcccc 120

ggaccttcaa aacctttttg ggttctcgtg gtggtgggcg gcgttctggc ctgctattct 180

ttgctggtaa ctgtagcctt cattatcttc tgggtt 216

<210> 268

<211> 771

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 268

gagagcaagt acggacctcc ttgtcctcca tgtcctgctc ccgagttcga gggcggacct 60

tcagtgttcc tgttcccccc taaacccaag gatactctta tgatcagccg gacccccgag 120

gtcacctgtg tggtggtaga tgttagccag gaggatcccg aggtgcagtt caactggtac 180

gtcgacggcg tcgaggtaca caacgccaag accaagccta gggaggagca gttccagtcc 240

acctataggg tcgtgagcgt gcttaccgtg ctgcaccagg actggttgaa cggcaaggag 300

tacaagtgca aggtgtccaa caagggcctc cccagcagca tcgagaagac cattagcaag 360

gcaaagggac agcccaggga gccccaggtg tacacattac ctccttccca ggaagagatg 420

accaagaacc aggtgtcgct tacctgcctg gtcaagggct tctacccctc cgacattgca 480

gttgaatggg agtcaaacgg ccagccggag aacaattaca agaccacccc cccagtcttg 540

gacagcgacg gctctttctt cctctactcg cggcttactg tagataaaag tcgttggcag 600

gagggaaacg tgttcagctg ctctgtgatg cacgaggccc tccataacca ctacacccag 660

aagagcctct ccctgtctct gggcaagatg ttctgggtgc tggtcgtggt gggcggagtt 720

cttgcttgct actccctgct cgtgaccgtc gctttcatta tattctgggt c 771

<210> 269

<211> 795

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 269

gagagaaagt gttgtgttga gtgtcctcct tgtcctccct gccctgctcc cgagttactt 60

ggcggacctt cagtgttcct gttccccccc aagcccaagg atactctcat gatcagccgg 120

acccccgagg tcacctgtgt ggtggtagat gttagccacg aggaccctga ggtcaagttc 180

aactggtacg tcgacggcgt cgaggtgcac aacgccaaga ccaagcctcg tgaagaacag 240

taccagtcca cctacagagt tgtgagcgtg cttaccgtgc tgcaccagga ctggctgaac 300

ggcaaggagt acaagtgcaa ggtgtccaac aaggccctcc ccgctcccat cgagaagaca 360

atcagcaagg ccaagccctg tccagcccct gagctcttag gaggacctag cgttttcctt 420

ttccctccca agcctaagga cactcttatg atctccagaa caccagaggt tacctgcgtc 480

gtggtggacg tgtcccatga ggacccagaa gtcaaattca attggtatgt agatggggtc 540

gaggtccaca acgctaagac aaagccccgc gaggagcagt acaactctac ctacagggtc 600

gtgtccgtgc tcacagtgct gcatcaggat tggctcaacg ggaaggagta taagtgcaaa 660

gtgtccaata aggcccttcc cgcccctatc gagaaaacca tctctaaggc caaattctgg 720

gtgctggtgg ttgtgggcgg cgtgcttgct tgttactccc tgctggtcac tgtagctttc 780

atcatatttt gggtg 795

<210> 270

<211> 494

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 270

agattcagcg ttgtgaagag aggccggaag aagctgctgt acatcttcaa gcagcccttc 60

atgagacctg tgcagaccac acaggaggaa gacggctgca gctgtagatt ccccgaggaa 120

gaggagggcg gctgtgagct gagagttaag ttcagcagga gcgccgacgc ccctgcctac 180

cagcaaggac agaatcaact gtacaacgag ctgaacctgg gcagacggga ggaatacgat 240

gtgctggaca agaggagagg cagagacccc gagatgggcg gcaaacctag aagaaagaac 300

ccccaggagg gcctgtataa cgagctccag aaggacaaga tggccgaggc ctacagcgag 360

atcggcatga agggcgaaag aagaagaggc aagggccacg acggcctcta ccagggctta 420

agcacagcta caaaggacac ctacgacgcc ctgcacatgc aggccctgcc ccctagatga 480

ttaattaaat cgat 494

<210> 271

<211> 1577

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 271

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca gcttcagctc caagagagcg gacctggctt agtgaagccc 120

agcgaaaccc tgtccctcac ctgcaccgtt tctggcggaa gcatcagcag ctccagctat 180

tactggggat ggatcaggca gccccctggc aagggtttag aatggatcgg ctcgatatat 240

tactccggca gcacctacta taaccccagc ttgaagagcc gggttaccat ttctgtggac 300

acatcaaaga accagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcggcatgg gctacggcat ggatgtgtgg 420

ggacaaggaa ccaccgttac tgtgagcagc ggttccacca gcggctcagg caagcctggc 480

tcaggagaag gaagcaccaa gggggatata cagatgacac agagcccctc cagcctgtcc 540

gccagcgttg gcgatcgtgt aacgatcacc tgccgggcct ctcagagcat caactcctac 600

ctcaattggt atcaacagaa gccaggcaag gcccccaaat tactcatcta cgccgccagc 660

agcttacaga gcggggttcc ctctagattc tccggctccg gttctggaac agatttcacc 720

ctcactatct ccagcttgca gcccgaggat ttcgccactt attactgtca gcagagcctg 780

gccgacccct tcacattcgg cggaggcaca aaggttgaga tcaaggcagc tgctttcgtg 840

cctgtgttcc tgcctgctaa gcccaccacc actcctgctc caagacctcc tacccccgct 900

cctacaatcg ccagccaacc tctgagcctg agaccggagg catgcagacc tgcggcaggg 960

ggagcagttc acacaagagg cttggacttc gcttgcgaca tctacatctg ggcccctctg 1020

gccggcacat gcggagttct tcttcttagc ctggtgatca ccctgtactg caaccacaga 1080

aacagattca gcgttgtgaa gagaggccgg aagaagctgc tgtacatctt caagcagccc 1140

ttcatgagac ctgtgcagac cacacaggag gaagacggct gcagctgtag attccccgag 1200

gaagaggagg gcggctgtga gctgagagtt aagttcagca ggagcgccga cgcccctgcc 1260

taccagcaag gacagaatca actgtacaac gagctgaacc tgggcagacg ggaggaatac 1320

gatgtgctgg acaagaggag aggcagagac cccgagatgg gcggcaaacc tagaagaaag 1380

aacccccagg agggcctgta taacgagctc cagaaggaca agatggccga ggcctacagc 1440

gagatcggca tgaagggcga aagaagaaga ggcaagggcc acgacggcct ctaccagggc 1500

ttaagcacag ctacaaagga cacctacgac gccctgcaca tgcaggccct gccccctaga 1560

tgattaatta aatcgat 1577

<210> 272

<211> 1577

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 272

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caagagagcg gacctggctt agtgaagccc 120

agcgaaaccc tgtccctcac ctgcaccgtt tctggcggaa gcatcagctc tcccgaccat 180

tactggggat ggatcaggca gccccctggc aagggtttgg aatggatcgg cagcatctac 240

gccagcggca gcacattcta caacccctcg ctcaaaagca gggttactat ttctgtggac 300

acaagcaaaa atcagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcgggatgg gctacggcat ggatgtgtgg 420

ggacaaggaa ccaccgttac tgtgagcagc ggctccacaa gcggctcagg caagcctggc 480

tcaggagaag gaagcaccaa gggggacatt caaatgaccc aaagcccctc cagcctgtcc 540

gccagcgttg gcgatagggt taccattacc tgcagggcca gccaaagcat caactcctac 600

ctaaattggt atcaacagaa gccaggcaag gcccccaaac tactcattta cgccgccagc 660

agcttacaga gcggggttcc ctctagattc tccggcagcg gttctggaac agatttcact 720

ctcacaatat cttcgctgca gcccgaggat ttcgctacct actattgcca gcaatccctg 780

gccgacccct tcacattcgg cggaggcaca aaggttgaga tcaaggcagc tgctttcgtg 840

cctgtgttcc tgcctgctaa gcccaccacc actcctgctc caagacctcc tacccccgct 900

cctacaatcg ccagccaacc tctgagcctg agaccggagg catgcagacc tgcggcaggg 960

ggagcagttc acacaagagg cttggacttc gcttgcgaca tctacatctg ggcccctctg 1020

gccggcacat gcggagttct tcttcttagc ctggtgatca ccctgtactg caaccacaga 1080

aacagattca gcgttgtgaa gagaggccgg aagaagctgc tgtacatctt caagcagccc 1140

ttcatgagac ctgtgcagac cacacaggag gaagacggct gcagctgtag attccccgag 1200

gaagaggagg gcggctgtga gctgagagtt aagttcagca ggagcgccga cgcccctgcc 1260

taccagcaag gacagaatca actgtacaac gagctgaacc tgggcagacg ggaggaatac 1320

gatgtgctgg acaagaggag aggcagagac cccgagatgg gcggcaaacc tagaagaaag 1380

aacccccagg agggcctgta taacgagctc cagaaggaca agatggccga ggcctacagc 1440

gagatcggca tgaagggcga aagaagaaga ggcaagggcc acgacggcct ctaccagggc 1500

ttaagcacag ctacaaagga cacctacgac gccctgcaca tgcaggccct gccccctaga 1560

tgattaatta aatcgat 1577

<210> 273

<211> 1574

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 273

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca gatcacatta aaagagagcg gacctacact ggtgaagccc 120

acccaaacgc ttaccctcac ctgcaccttt agcgggttca gcctggacac agagggcgtt 180

ggcgttggat ggatcaggca gcctcctggc aaagccctcg aatggcttgc cctcatctac 240

ttcaacgacc agaagagata cagcccctcc ttaaaatctc ggctcacaat caccaaagac 300

acaagcaaaa atcaggttgt gctcaccatg accaacatgg accctgtgga caccgctgtg 360

tactactgtg ccagagacac cggctacagc agatggtact acgggatgga cgtttggggc 420

caaggcacca ctgtgaccgt ttccagcggc tctacaagcg gcagcgggaa acctggttct 480

ggagagggca gcacaaaggg cgacatccag atgacgcaat cccccagctc tgtgagcgcc 540

tctgtgggag acagagttac aatcacatgc cgggcctccc agggcatcag ctcttggctg 600

gcatggtatc aacagaagcc tggcaaggct cccaagctgc tcatctatgc cgcctcctcc 660

ttacaatctg gagttccctc caggttcagc gggagcggct caggaacaga cttcaccctt 720

accatctcta gcctgcaacc cgaggacttc gctacttatt actgtcagca ggcctacgcc 780

taccccatca cattcggcgg aggaacaaag gttgagatca aggcagctgc tttcgtgcct 840

gtgttcctgc ctgctaagcc caccaccact cctgctccaa gacctcctac ccccgctcct 900

acaatcgcca gccaacctct gagcctgaga ccggaggcat gcagacctgc ggcaggggga 960

gcagttcaca caagaggctt ggacttcgct tgcgacatct acatctgggc ccctctggcc 1020

ggcacatgcg gagttcttct tcttagcctg gtgatcaccc tgtactgcaa ccacagaaac 1080

agattcagcg ttgtgaagag aggccggaag aagctgctgt acatcttcaa gcagcccttc 1140

atgagacctg tgcagaccac acaggaggaa gacggctgca gctgtagatt ccccgaggaa 1200

gaggagggcg gctgtgagct gagagttaag ttcagcagga gcgccgacgc ccctgcctac 1260

cagcaaggac agaatcaact gtacaacgag ctgaacctgg gcagacggga ggaatacgat 1320

gtgctggaca agaggagagg cagagacccc gagatgggcg gcaaacctag aagaaagaac 1380

ccccaggagg gcctgtataa cgagctccag aaggacaaga tggccgaggc ctacagcgag 1440

atcggcatga agggcgaaag aagaagaggc aagggccacg acggcctcta ccagggctta 1500

agcacagcta caaaggacac ctacgacgcc ctgcacatgc aggccctgcc ccctagatga 1560

ttaattaaat cgat 1574

<210> 274

<211> 1592

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 274

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttccagaa atactactgg 180

agctggatcc ggcagcctcc cggcaaaggc ttagaatgga tcggagagat agacaccagc 240

ggcttcacca actacaaccc cagcctgaaa tctagggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttggcagata cagctacggc tactacatca ccgccttcga catttggggc 420

caaggcacca ctgtgaccgt ttccagcgga agcactagcg gcagcgggaa acctggttct 480

ggagagggct caaccaaggg cgacatcgtg atgacacaga gccccgactc tctggctgtg 540

tccctgggag agagagccac catcaactgc aagagcagcc agagcgttct gtacagcagc 600

aacaacaaga actacctggc atggtatcaa cagaagcctg gccagccccc taagctgctc 660

atctactggg cttccaccag agaatcaggc gttccagaca ggttctccgg ctcgggttca 720

ggcacagact tcacccttac catctcttcc ctgcaggccg aagatgtggc cgtttactac 780

tgtcagcagc actacagctt ccctttcaca ttcggcggag gcaccaaggt tgagatcaag 840

gcagctgctt tcgtgcctgt gttcctgcct gctaagccca ccaccactcc tgctccaaga 900

cctcctaccc ccgctcctac aatcgccagc caacctctga gcctgagacc ggaggcatgc 960

agacctgcgg cagggggagc agttcacaca agaggcttgg acttcgcttg cgacatctac 1020

atctgggccc ctctggccgg cacatgcgga gttcttcttc ttagcctggt gatcaccctg 1080

tactgcaacc acagaaacag attcagcgtt gtgaagagag gccggaagaa gctgctgtac 1140

atcttcaagc agcccttcat gagacctgtg cagaccacac aggaggaaga cggctgcagc 1200

tgtagattcc ccgaggaaga ggagggcggc tgtgagctga gagttaagtt cagcaggagc 1260

gccgacgccc ctgcctacca gcaaggacag aatcaactgt acaacgagct gaacctgggc 1320

agacgggagg aatacgatgt gctggacaag aggagaggca gagaccccga gatgggcggc 1380

aaacctagaa gaaagaaccc ccaggagggc ctgtataacg agctccagaa ggacaagatg 1440

gccgaggcct acagcgagat cggcatgaag ggcgaaagaa gaagaggcaa gggccacgac 1500

ggcctctacc agggcttaag cacagctaca aaggacacct acgacgccct gcacatgcag 1560

gccctgcccc ctagatgatt aattaaatcg at 1592

<210> 275

<211> 1604

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 275

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcgagaa atactactgg 180

agctggatcc ggcagcctcc cggcaaaggc ttagaatgga tcggcgagat ttatcacagc 240

gggctcacca actacaaccc cagcctgaaa tctcgagtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttact gtctcctctg gatctaccag cggcagcggc 480

aagcctggat ctggcgaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggctg tgtctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcttggtatc aacagaagcc tggccagccc 660

cctaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tcggtttagc 720

ggctccggct caggaaccga tttcaccctc actatctcca gcctccaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca aggcagctgc tttcgtgcct gtgttcctgc ctgctaagcc caccaccact 900

cctgctccaa gacctcctac ccccgctcct acaatcgcca gccaacctct gagcctgaga 960

ccggaggcat gcagacctgc ggcaggggga gcagttcaca caagaggctt ggacttcgct 1020

tgcgacatct acatctgggc ccctctggcc ggcacatgcg gagttcttct tcttagcctg 1080

gtgatcaccc tgtactgcaa ccacagaaac agattcagcg ttgtgaagag aggccggaag 1140

aagctgctgt acatcttcaa gcagcccttc atgagacctg tgcagaccac acaggaggaa 1200

gacggctgca gctgtagatt ccccgaggaa gaggagggcg gctgtgagct gagagttaag 1260

ttcagcagga gcgccgacgc ccctgcctac cagcaaggac agaatcaact gtacaacgag 1320

ctgaacctgg gcagacggga ggaatacgat gtgctggaca agaggagagg cagagacccc 1380

gagatgggcg gcaaacctag aagaaagaac ccccaggagg gcctgtataa cgagctccag 1440

aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgaaag aagaagaggc 1500

aagggccacg acggcctcta ccagggctta agcacagcta caaaggacac ctacgacgcc 1560

ctgcacatgc aggccctgcc ccctagatga ttaattaaat cgat 1604

<210> 276

<211> 1604

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 276

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagccg ctatgtgtgg 180

agctggatcc ggcagcctcc tggcaaaggc cttgaatgga tcggagagat agacagcagc 240

ggcaagacca actacaaccc cagcctgaaa tcacgcgtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagctccgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttaca gttagctctg gaagcaccag cggctccggc 480

aagcctggat ctggtgaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggctg tgtctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcatggtatc aacagaagcc tggccagcct 660

cccaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tcgctttagc 720

ggcagcggtt ctggcaccga tttcactctt acaatcagca gcttacaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca aggcagctgc tttcgtgcct gtgttcctgc ctgctaagcc caccaccact 900

cctgctccaa gacctcctac ccccgctcct acaatcgcca gccaacctct gagcctgaga 960

ccggaggcat gcagacctgc ggcaggggga gcagttcaca caagaggctt ggacttcgct 1020

tgcgacatct acatctgggc ccctctggcc ggcacatgcg gagttcttct tcttagcctg 1080

gtgatcaccc tgtactgcaa ccacagaaac agattcagcg ttgtgaagag aggccggaag 1140

aagctgctgt acatcttcaa gcagcccttc atgagacctg tgcagaccac acaggaggaa 1200

gacggctgca gctgtagatt ccccgaggaa gaggagggcg gctgtgagct gagagttaag 1260

ttcagcagga gcgccgacgc ccctgcctac cagcaaggac agaatcaact gtacaacgag 1320

ctgaacctgg gcagacggga ggaatacgat gtgctggaca agaggagagg cagagacccc 1380

gagatgggcg gcaaacctag aagaaagaac ccccaggagg gcctgtataa cgagctccag 1440

aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgaaag aagaagaggc 1500

aagggccacg acggcctcta ccagggctta agcacagcta caaaggacac ctacgacgcc 1560

ctgcacatgc aggccctgcc ccctagatga ttaattaaat cgat 1604

<210> 277

<211> 1604

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 277

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg ctacgcttgg 180

agctggatta gacagcctcc tggcaaagga ctagaatgga tcggagagat cgaccacaga 240

ggcttcacca actacaaccc cagcctgaaa tccagagtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccaggg ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttacg gttagctctg gatctaccag cggcagcggc 480

aagcctggct caggagaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggccg tttctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcatggtatc aacagaagcc aggccagcct 660

cccaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tagattttcg 720

ggatcaggct ccggcaccga tttcactctt acgatcagca gcttacaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca aggcagctgc tttcgtgcct gtgttcctgc ctgctaagcc caccaccact 900

cctgctccaa gacctcctac ccccgctcct acaatcgcca gccaacctct gagcctgaga 960

ccggaggcat gcagacctgc ggcaggggga gcagttcaca caagaggctt ggacttcgct 1020

tgcgacatct acatctgggc ccctctggcc ggcacatgcg gagttcttct tcttagcctg 1080

gtgatcaccc tgtactgcaa ccacagaaac agattcagcg ttgtgaagag aggccggaag 1140

aagctgctgt acatcttcaa gcagcccttc atgagacctg tgcagaccac acaggaggaa 1200

gacggctgca gctgtagatt ccccgaggaa gaggagggcg gctgtgagct gagagttaag 1260

ttcagcagga gcgccgacgc ccctgcctac cagcaaggac agaatcaact gtacaacgag 1320

ctgaacctgg gcagacggga ggaatacgat gtgctggaca agaggagagg cagagacccc 1380

gagatgggcg gcaaacctag aagaaagaac ccccaggagg gcctgtataa cgagctccag 1440

aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgaaag aagaagaggc 1500

aagggccacg acggcctcta ccagggctta agcacagcta caaaggacac ctacgacgcc 1560

ctgcacatgc aggccctgcc ccctagatga ttaattaaat cgat 1604

<210> 278

<211> 1565

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 278

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg ctattactgg 180

agctggatcc ggcagcctcc tggaaaagga ttagaatgga tcggcgagat agaccacagc 240

gggagcacaa actacaaccc cagcctgaaa tcgcgggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag gcggaggctc ctggtacagc aactggttcg atccttgggg ccaaggcacc 420

atggtgaccg tttccagcgg ctctacaagc ggcagcggga aacctggttc tggagagggc 480

agcacaaagg gcgacatcca gatgacacag agccccagca cccttagcgc ctctgtggga 540

gatagggtta ccattacctg cagggcttcc cagagcatca gcagctggct ggcatggtat 600

caacagaagc ctggcaaggc tcccaagctg ctcatctatg acgcctccag cctggaaagc 660

ggggttccct ccagatttag cggctcaggc tccggaacag agttcaccct taccatctct 720

agcctgcaac ccgacgactt cgctacttat tactgtcaac aagacagaag cttgcccccc 780

acattcggcg gagggaccaa ggttgagatc aaggcagctg ctttcgtgcc tgtgttcctg 840

cctgctaagc ccaccaccac tcctgctcca agacctccta cccccgctcc tacaatcgcc 900

agccaacctc tgagcctgag accggaggca tgcagacctg cggcaggggg agcagttcac 960

acaagaggct tggacttcgc ttgcgacatc tacatctggg cccctctggc cggcacatgc 1020

ggagttcttc ttcttagcct ggtgatcacc ctgtactgca accacagaaa cagattcagc 1080

gttgtgaaga gaggccggaa gaagctgctg tacatcttca agcagccctt catgagacct 1140

gtgcagacca cacaggagga agacggctgc agctgtagat tccccgagga agaggagggc 1200

ggctgtgagc tgagagttaa gttcagcagg agcgccgacg cccctgccta ccagcaagga 1260

cagaatcaac tgtacaacga gctgaacctg ggcagacggg aggaatacga tgtgctggac 1320

aagaggagag gcagagaccc cgagatgggc ggcaaaccta gaagaaagaa cccccaggag 1380

ggcctgtata acgagctcca gaaggacaag atggccgagg cctacagcga gatcggcatg 1440

aagggcgaaa gaagaagagg caagggccac gacggcctct accagggctt aagcacagct 1500

acaaaggaca cctacgacgc cctgcacatg caggccctgc cccctagatg attaattaaa 1560

tcgat 1565

<210> 279

<211> 1580

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 279

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagctt gtgcagagcg gagctgaagt taagaagcct 120

ggcgcctctg tgaaggttag ctgcaaggcc agcggctaca cattcaagga atatggcatc 180

tcctgggtta ggcaggctcc cggccaaggc ttagaatgga tgggctggat ctccgcctac 240

tccggccaca cctactacgc ccagaagctt cagggcaggg ttaccatgac caccgacacc 300

agcacctcta ccgcctatat ggagctgagg agcctgagat cggacgacac agctgtgtat 360

tactgcgcca gaggccccca ctacgacgac tggtctggat ttatcatctg gttcgacccc 420

tgggggcagg gcaccctggt cacagtttct tctggctcca ccagcggaag cggcaagcca 480

ggctcaggcg aaggatctac aaaaggcgac atccaaatga cacagagccc cagcagcttg 540

agcgcctccg ttggcgacag agttacaatc acctgcaggg cctctcagag catcagcagc 600

tatttgaatt ggtatcaaca gaagccagga aaggccccta agctgctcat ctacgctgcc 660

agctcgctcc aatctggcgt tcctagcaga tttagcggct ccggcagcgg cacagacttt 720

actcttacca ttagctccct gcagcccgag gacttcgcta cctactattg ccagcaaagc 780

tacagattcc ctcccacctt tggccagggc acaaaggttg agatcaaggc agctgctttc 840

gtgcctgtgt tcctgcctgc taagcccacc accactcctg ctccaagacc tcctaccccc 900

gctcctacaa tcgccagcca acctctgagc ctgagaccgg aggcatgcag acctgcggca 960

gggggagcag ttcacacaag aggcttggac ttcgcttgcg acatctacat ctgggcccct 1020

ctggccggca catgcggagt tcttcttctt agcctggtga tcaccctgta ctgcaaccac 1080

agaaacagat tcagcgttgt gaagagaggc cggaagaagc tgctgtacat cttcaagcag 1140

cccttcatga gacctgtgca gaccacacag gaggaagacg gctgcagctg tagattcccc 1200

gaggaagagg agggcggctg tgagctgaga gttaagttca gcaggagcgc cgacgcccct 1260

gcctaccagc aaggacagaa tcaactgtac aacgagctga acctgggcag acgggaggaa 1320

tacgatgtgc tggacaagag gagaggcaga gaccccgaga tgggcggcaa acctagaaga 1380

aagaaccccc aggagggcct gtataacgag ctccagaagg acaagatggc cgaggcctac 1440

agcgagatcg gcatgaaggg cgaaagaaga agaggcaagg gccacgacgg cctctaccag 1500

ggcttaagca cagctacaaa ggacacctac gacgccctgc acatgcaggc cctgccccct 1560

agatgattaa ttaaatcgat 1580

<210> 280

<211> 1586

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 280

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg catccactgg 180

aactggatcc ggcagcctcc tggcaaaggc cttgaatgga tcggcgatat cgacaccagc 240

ggctccacca actacaaccc cagcctgaaa tcgagggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagac tgggccagga aagcgctacc taccttggca tggatgtgtg ggggcagggc 420

accaccgtta ctgttagctc tggctcaaca agcggcagcg gcaagcctgg ctcaggagaa 480

ggaagcacaa agggcgacat tgtaatgact cagagccccg acagcctggc cgttagctta 540

ggcgaaaggg ctacaatcaa ttgcaagagc agccagagcg ttctgtacag cagcaacaac 600

aagaactacc tcgcatggta tcaacagaag ccaggccagc ctcccaagct gctcatctac 660

tgggcttcca ccagagagag cggggttccc gatagattct ccggctccgg ttctggaaca 720

gatttcacgc tcacaatcag cagcttacag gccgaggatg tggctgtcta ctattgtcag 780

cagttgtaca cctacccctt cacattcggc ggaggcacca aggttgagat caaggcagct 840

gctttcgtgc ctgtgttcct gcctgctaag cccaccacca ctcctgctcc aagacctcct 900

acccccgctc ctacaatcgc cagccaacct ctgagcctga gaccggaggc atgcagacct 960

gcggcagggg gagcagttca cacaagaggc ttggacttcg cttgcgacat ctacatctgg 1020

gcccctctgg ccggcacatg cggagttctt cttcttagcc tggtgatcac cctgtactgc 1080

aaccacagaa acagattcag cgttgtgaag agaggccgga agaagctgct gtacatcttc 1140

aagcagccct tcatgagacc tgtgcagacc acacaggagg aagacggctg cagctgtaga 1200

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1260

gcccctgcct accagcaagg acagaatcaa ctgtacaacg agctgaacct gggcagacgg 1320

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1380

agaagaaaga acccccagga gggcctgtat aacgagctcc agaaggacaa gatggccgag 1440

gcctacagcg agatcggcat gaagggcgaa agaagaagag gcaagggcca cgacggcctc 1500

taccagggct taagcacagc tacaaaggac acctacgacg ccctgcacat gcaggccctg 1560

ccccctagat gattaattaa atcgat 1586

<210> 281

<211> 1499

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 281

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca gcttcagctc caagagagcg gacctggctt agtgaagccc 120

agcgaaaccc tgtccctcac ctgcaccgtt tctggcggaa gcatcagcag ctccagctat 180

tactggggat ggatcaggca gccccctggc aagggtttag aatggatcgg ctcgatatat 240

tactccggca gcacctacta taaccccagc ttgaagagcc gggttaccat ttctgtggac 300

acatcaaaga accagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcggcatgg gctacggcat ggatgtgtgg 420

ggacaaggaa ccaccgttac tgtgagcagc ggttccacca gcggctcagg caagcctggc 480

tcaggagaag gaagcaccaa gggggatata cagatgacac agagcccctc cagcctgtcc 540

gccagcgttg gcgatcgtgt aacgatcacc tgccgggcct ctcagagcat caactcctac 600

ctcaattggt atcaacagaa gccaggcaag gcccccaaat tactcatcta cgccgccagc 660

agcttacaga gcggggttcc ctctagattc tccggctccg gttctggaac agatttcacc 720

ctcactatct ccagcttgca gcccgaggat ttcgccactt attactgtca gcagagcctg 780

gccgacccct tcacattcgg cggaggcaca aaggttgaga tcaaggctgc tgcattggat 840

aatgaaaaat cgaacggcac aatcattcat gtgaagggca aacacctgtg tcccagcccc 900

ttgttcccag gacctagcaa gcctttttgg gttctcgtgg tggtgggcgg cgttctggct 960

tgctactctc tacttgtaac tgtcgcattt attatattct gggttagatt cagcgttgtg 1020

aagagaggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgag acctgtgcag 1080

accacacagg aggaagacgg ctgcagctgt agattccccg aggaagagga gggcggctgt 1140

gagctgagag ttaagttcag caggagcgcc gacgcccctg cctaccagca aggacagaat 1200

caactgtaca acgagctgaa cctgggcaga cgggaggaat acgatgtgct ggacaagagg 1260

agaggcagag accccgagat gggcggcaaa cctagaagaa agaaccccca ggagggcctg 1320

tataacgagc tccagaagga caagatggcc gaggcctaca gcgagatcgg catgaagggc 1380

gaaagaagaa gaggcaaggg ccacgacggc ctctaccagg gcttaagcac agctacaaag 1440

gacacctacg acgccctgca catgcaggcc ctgcccccta gatgattaat taaatcgat 1499

<210> 282

<211> 1499

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 282

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caagagagcg gacctggctt agtgaagccc 120

agcgaaaccc tgtccctcac ctgcaccgtt tctggcggaa gcatcagctc tcccgaccat 180

tactggggat ggatcaggca gccccctggc aagggtttgg aatggatcgg cagcatctac 240

gccagcggca gcacattcta caacccctcg ctcaaaagca gggttactat ttctgtggac 300

acaagcaaaa atcagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcgggatgg gctacggcat ggatgtgtgg 420

ggacaaggaa ccaccgttac tgtgagcagc ggctccacaa gcggctcagg caagcctggc 480

tcaggagaag gaagcaccaa gggggacatt caaatgaccc aaagcccctc cagcctgtcc 540

gccagcgttg gcgatagggt taccattacc tgcagggcca gccaaagcat caactcctac 600

ctaaattggt atcaacagaa gccaggcaag gcccccaaac tactcattta cgccgccagc 660

agcttacaga gcggggttcc ctctagattc tccggcagcg gttctggaac agatttcact 720

ctcacaatat cttcgctgca gcccgaggat ttcgctacct actattgcca gcaatccctg 780

gccgacccct tcacattcgg cggaggcaca aaggttgaga tcaaggctgc tgcattggat 840

aatgaaaaat cgaacggcac aatcattcat gtgaagggca aacacctgtg tcccagcccc 900

ttgttcccag gacctagcaa gcctttttgg gttctcgtgg tggtgggcgg cgttctggct 960

tgctactctc tacttgtaac tgtcgcattt attatattct gggttagatt cagcgttgtg 1020

aagagaggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgag acctgtgcag 1080

accacacagg aggaagacgg ctgcagctgt agattccccg aggaagagga gggcggctgt 1140

gagctgagag ttaagttcag caggagcgcc gacgcccctg cctaccagca aggacagaat 1200

caactgtaca acgagctgaa cctgggcaga cgggaggaat acgatgtgct ggacaagagg 1260

agaggcagag accccgagat gggcggcaaa cctagaagaa agaaccccca ggagggcctg 1320

tataacgagc tccagaagga caagatggcc gaggcctaca gcgagatcgg catgaagggc 1380

gaaagaagaa gaggcaaggg ccacgacggc ctctaccagg gcttaagcac agctacaaag 1440

gacacctacg acgccctgca catgcaggcc ctgcccccta gatgattaat taaatcgat 1499

<210> 283

<211> 1496

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 283

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca gatcacatta aaagagagcg gacctacact ggtgaagccc 120

acccaaacgc ttaccctcac ctgcaccttt agcgggttca gcctggacac agagggcgtt 180

ggcgttggat ggatcaggca gcctcctggc aaagccctcg aatggcttgc cctcatctac 240

ttcaacgacc agaagagata cagcccctcc ttaaaatctc ggctcacaat caccaaagac 300

acaagcaaaa atcaggttgt gctcaccatg accaacatgg accctgtgga caccgctgtg 360

tactactgtg ccagagacac cggctacagc agatggtact acgggatgga cgtttggggc 420

caaggcacca ctgtgaccgt ttccagcggc tctacaagcg gcagcgggaa acctggttct 480

ggagagggca gcacaaaggg cgacatccag atgacgcaat cccccagctc tgtgagcgcc 540

tctgtgggag acagagttac aatcacatgc cgggcctccc agggcatcag ctcttggctg 600

gcatggtatc aacagaagcc tggcaaggct cccaagctgc tcatctatgc cgcctcctcc 660

ttacaatctg gagttccctc caggttcagc gggagcggct caggaacaga cttcaccctt 720

accatctcta gcctgcaacc cgaggacttc gctacttatt actgtcagca ggcctacgcc 780

taccccatca cattcggcgg aggaacaaag gttgagatca aggctgctgc attggataat 840

gaaaaatcga acggcacaat cattcatgtg aagggcaaac acctgtgtcc cagccccttg 900

ttcccaggac ctagcaagcc tttttgggtt ctcgtggtgg tgggcggcgt tctggcttgc 960

tactctctac ttgtaactgt cgcatttatt atattctggg ttagattcag cgttgtgaag 1020

agaggccgga agaagctgct gtacatcttc aagcagccct tcatgagacc tgtgcagacc 1080

acacaggagg aagacggctg cagctgtaga ttccccgagg aagaggaggg cggctgtgag 1140

ctgagagtta agttcagcag gagcgccgac gcccctgcct accagcaagg acagaatcaa 1200

ctgtacaacg agctgaacct gggcagacgg gaggaatacg atgtgctgga caagaggaga 1260

ggcagagacc ccgagatggg cggcaaacct agaagaaaga acccccagga gggcctgtat 1320

aacgagctcc agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgaa 1380

agaagaagag gcaagggcca cgacggcctc taccagggct taagcacagc tacaaaggac 1440

acctacgacg ccctgcacat gcaggccctg ccccctagat gattaattaa atcgat 1496

<210> 284

<211> 1514

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 284

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttccagaa atactactgg 180

agctggatcc ggcagcctcc cggcaaaggc ttagaatgga tcggagagat agacaccagc 240

ggcttcacca actacaaccc cagcctgaaa tctagggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttggcagata cagctacggc tactacatca ccgccttcga catttggggc 420

caaggcacca ctgtgaccgt ttccagcgga agcactagcg gcagcgggaa acctggttct 480

ggagagggct caaccaaggg cgacatcgtg atgacacaga gccccgactc tctggctgtg 540

tccctgggag agagagccac catcaactgc aagagcagcc agagcgttct gtacagcagc 600

aacaacaaga actacctggc atggtatcaa cagaagcctg gccagccccc taagctgctc 660

atctactggg cttccaccag agaatcaggc gttccagaca ggttctccgg ctcgggttca 720

ggcacagact tcacccttac catctcttcc ctgcaggccg aagatgtggc cgtttactac 780

tgtcagcagc actacagctt ccctttcaca ttcggcggag gcaccaaggt tgagatcaag 840

gctgctgcat tggataatga aaaatcgaac ggcacaatca ttcatgtgaa gggcaaacac 900

ctgtgtccca gccccttgtt cccaggacct agcaagcctt tttgggttct cgtggtggtg 960

ggcggcgttc tggcttgcta ctctctactt gtaactgtcg catttattat attctgggtt 1020

agattcagcg ttgtgaagag aggccggaag aagctgctgt acatcttcaa gcagcccttc 1080

atgagacctg tgcagaccac acaggaggaa gacggctgca gctgtagatt ccccgaggaa 1140

gaggagggcg gctgtgagct gagagttaag ttcagcagga gcgccgacgc ccctgcctac 1200

cagcaaggac agaatcaact gtacaacgag ctgaacctgg gcagacggga ggaatacgat 1260

gtgctggaca agaggagagg cagagacccc gagatgggcg gcaaacctag aagaaagaac 1320

ccccaggagg gcctgtataa cgagctccag aaggacaaga tggccgaggc ctacagcgag 1380

atcggcatga agggcgaaag aagaagaggc aagggccacg acggcctcta ccagggctta 1440

agcacagcta caaaggacac ctacgacgcc ctgcacatgc aggccctgcc ccctagatga 1500

ttaattaaat cgat 1514

<210> 285

<211> 1526

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 285

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcgagaa atactactgg 180

agctggatcc ggcagcctcc cggcaaaggc ttagaatgga tcggcgagat ttatcacagc 240

gggctcacca actacaaccc cagcctgaaa tctcgagtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttact gtctcctctg gatctaccag cggcagcggc 480

aagcctggat ctggcgaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggctg tgtctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcttggtatc aacagaagcc tggccagccc 660

cctaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tcggtttagc 720

ggctccggct caggaaccga tttcaccctc actatctcca gcctccaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca aggctgctgc attggataat gaaaaatcga acggcacaat cattcatgtg 900

aagggcaaac acctgtgtcc cagccccttg ttcccaggac ctagcaagcc tttttgggtt 960

ctcgtggtgg tgggcggcgt tctggcttgc tactctctac ttgtaactgt cgcatttatt 1020

atattctggg ttagattcag cgttgtgaag agaggccgga agaagctgct gtacatcttc 1080

aagcagccct tcatgagacc tgtgcagacc acacaggagg aagacggctg cagctgtaga 1140

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1200

gcccctgcct accagcaagg acagaatcaa ctgtacaacg agctgaacct gggcagacgg 1260

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1320

agaagaaaga acccccagga gggcctgtat aacgagctcc agaaggacaa gatggccgag 1380

gcctacagcg agatcggcat gaagggcgaa agaagaagag gcaagggcca cgacggcctc 1440

taccagggct taagcacagc tacaaaggac acctacgacg ccctgcacat gcaggccctg 1500

ccccctagat gattaattaa atcgat 1526

<210> 286

<211> 1526

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 286

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagccg ctatgtgtgg 180

agctggatcc ggcagcctcc tggcaaaggc cttgaatgga tcggagagat agacagcagc 240

ggcaagacca actacaaccc cagcctgaaa tcacgcgtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagctccgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttaca gttagctctg gaagcaccag cggctccggc 480

aagcctggat ctggtgaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggctg tgtctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcatggtatc aacagaagcc tggccagcct 660

cccaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tcgctttagc 720

ggcagcggtt ctggcaccga tttcactctt acaatcagca gcttacaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca aggctgctgc attggataat gaaaaatcga acggcacaat cattcatgtg 900

aagggcaaac acctgtgtcc cagccccttg ttcccaggac ctagcaagcc tttttgggtt 960

ctcgtggtgg tgggcggcgt tctggcttgc tactctctac ttgtaactgt cgcatttatt 1020

atattctggg ttagattcag cgttgtgaag agaggccgga agaagctgct gtacatcttc 1080

aagcagccct tcatgagacc tgtgcagacc acacaggagg aagacggctg cagctgtaga 1140

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1200

gcccctgcct accagcaagg acagaatcaa ctgtacaacg agctgaacct gggcagacgg 1260

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1320

agaagaaaga acccccagga gggcctgtat aacgagctcc agaaggacaa gatggccgag 1380

gcctacagcg agatcggcat gaagggcgaa agaagaagag gcaagggcca cgacggcctc 1440

taccagggct taagcacagc tacaaaggac acctacgacg ccctgcacat gcaggccctg 1500

ccccctagat gattaattaa atcgat 1526

<210> 287

<211> 1526

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 287

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagtta caacaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg ctacgcttgg 180

agctggatta gacagcctcc tggcaaagga ctagaatgga tcggagagat cgaccacaga 240

ggcttcacca actacaaccc cagcctgaaa tccagagtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccaggg ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttacg gttagctctg gatctaccag cggcagcggc 480

aagcctggct caggagaagg aagcacaaag ggcgacattg tgctcaccca gagccccgac 540

agcctggccg tttctttagg cgaaagggct accatcaact gcaagagcag ccagagcgtt 600

ctgtacagca gcaacaacaa gaactacctt gcatggtatc aacagaagcc aggccagcct 660

cccaagctgc tcatctactg ggcctctagc agagagagcg gggttcccga tagattttcg 720

ggatcaggct ccggcaccga tttcactctt acgatcagca gcttacaggc cgaggatgtg 780

gctgtctact attgtcagca gagctatagc ttcccctgga cattcggcgg aggcaccaag 840

gttgagatca aggctgctgc attggataat gaaaaatcga acggcacaat cattcatgtg 900

aagggcaaac acctgtgtcc cagccccttg ttcccaggac ctagcaagcc tttttgggtt 960

ctcgtggtgg tgggcggcgt tctggcttgc tactctctac ttgtaactgt cgcatttatt 1020

atattctggg ttagattcag cgttgtgaag agaggccgga agaagctgct gtacatcttc 1080

aagcagccct tcatgagacc tgtgcagacc acacaggagg aagacggctg cagctgtaga 1140

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1200

gcccctgcct accagcaagg acagaatcaa ctgtacaacg agctgaacct gggcagacgg 1260

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1320

agaagaaaga acccccagga gggcctgtat aacgagctcc agaaggacaa gatggccgag 1380

gcctacagcg agatcggcat gaagggcgaa agaagaagag gcaagggcca cgacggcctc 1440

taccagggct taagcacagc tacaaaggac acctacgacg ccctgcacat gcaggccctg 1500

ccccctagat gattaattaa atcgat 1526

<210> 288

<211> 1487

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 288

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg ctattactgg 180

agctggatcc ggcagcctcc tggaaaagga ttagaatgga tcggcgagat agaccacagc 240

gggagcacaa actacaaccc cagcctgaaa tcgcgggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagag gcggaggctc ctggtacagc aactggttcg atccttgggg ccaaggcacc 420

atggtgaccg tttccagcgg ctctacaagc ggcagcggga aacctggttc tggagagggc 480

agcacaaagg gcgacatcca gatgacacag agccccagca cccttagcgc ctctgtggga 540

gatagggtta ccattacctg cagggcttcc cagagcatca gcagctggct ggcatggtat 600

caacagaagc ctggcaaggc tcccaagctg ctcatctatg acgcctccag cctggaaagc 660

ggggttccct ccagatttag cggctcaggc tccggaacag agttcaccct taccatctct 720

agcctgcaac ccgacgactt cgctacttat tactgtcaac aagacagaag cttgcccccc 780

acattcggcg gagggaccaa ggttgagatc aaggctgctg cattggataa tgaaaaatcg 840

aacggcacaa tcattcatgt gaagggcaaa cacctgtgtc ccagcccctt gttcccagga 900

cctagcaagc ctttttgggt tctcgtggtg gtgggcggcg ttctggcttg ctactctcta 960

cttgtaactg tcgcatttat tatattctgg gttagattca gcgttgtgaa gagaggccgg 1020

aagaagctgc tgtacatctt caagcagccc ttcatgagac ctgtgcagac cacacaggag 1080

gaagacggct gcagctgtag attccccgag gaagaggagg gcggctgtga gctgagagtt 1140

aagttcagca ggagcgccga cgcccctgcc taccagcaag gacagaatca actgtacaac 1200

gagctgaacc tgggcagacg ggaggaatac gatgtgctgg acaagaggag aggcagagac 1260

cccgagatgg gcggcaaacc tagaagaaag aacccccagg agggcctgta taacgagctc 1320

cagaaggaca agatggccga ggcctacagc gagatcggca tgaagggcga aagaagaaga 1380

ggcaagggcc acgacggcct ctaccagggc ttaagcacag ctacaaagga cacctacgac 1440

gccctgcaca tgcaggccct gccccctaga tgattaatta aatcgat 1487

<210> 289

<211> 1502

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 289

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagctt gtgcagagcg gagctgaagt taagaagcct 120

ggcgcctctg tgaaggttag ctgcaaggcc agcggctaca cattcaagga atatggcatc 180

tcctgggtta ggcaggctcc cggccaaggc ttagaatgga tgggctggat ctccgcctac 240

tccggccaca cctactacgc ccagaagctt cagggcaggg ttaccatgac caccgacacc 300

agcacctcta ccgcctatat ggagctgagg agcctgagat cggacgacac agctgtgtat 360

tactgcgcca gaggccccca ctacgacgac tggtctggat ttatcatctg gttcgacccc 420

tgggggcagg gcaccctggt cacagtttct tctggctcca ccagcggaag cggcaagcca 480

ggctcaggcg aaggatctac aaaaggcgac atccaaatga cacagagccc cagcagcttg 540

agcgcctccg ttggcgacag agttacaatc acctgcaggg cctctcagag catcagcagc 600

tatttgaatt ggtatcaaca gaagccagga aaggccccta agctgctcat ctacgctgcc 660

agctcgctcc aatctggcgt tcctagcaga tttagcggct ccggcagcgg cacagacttt 720

actcttacca ttagctccct gcagcccgag gacttcgcta cctactattg ccagcaaagc 780

tacagattcc ctcccacctt tggccagggc acaaaggttg agatcaaggc tgctgcattg 840

gataatgaaa aatcgaacgg cacaatcatt catgtgaagg gcaaacacct gtgtcccagc 900

cccttgttcc caggacctag caagcctttt tgggttctcg tggtggtggg cggcgttctg 960

gcttgctact ctctacttgt aactgtcgca tttattatat tctgggttag attcagcgtt 1020

gtgaagagag gccggaagaa gctgctgtac atcttcaagc agcccttcat gagacctgtg 1080

cagaccacac aggaggaaga cggctgcagc tgtagattcc ccgaggaaga ggagggcggc 1140

tgtgagctga gagttaagtt cagcaggagc gccgacgccc ctgcctacca gcaaggacag 1200

aatcaactgt acaacgagct gaacctgggc agacgggagg aatacgatgt gctggacaag 1260

aggagaggca gagaccccga gatgggcggc aaacctagaa gaaagaaccc ccaggagggc 1320

ctgtataacg agctccagaa ggacaagatg gccgaggcct acagcgagat cggcatgaag 1380

ggcgaaagaa gaagaggcaa gggccacgac ggcctctacc agggcttaag cacagctaca 1440

aaggacacct acgacgccct gcacatgcag gccctgcccc ctagatgatt aattaaatcg 1500

at 1502

<210> 290

<211> 1508

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 290

ggtacccccg ggcccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt 60

ctgcatgctg ctagacctca ggttcagttg cagcaatggg gagctggcct gttaaagccc 120

agcgaaaccc tgtccctcac ctgcgctgtg tatggcggaa gcttcagcgg catccactgg 180

aactggatcc ggcagcctcc tggcaaaggc cttgaatgga tcggcgatat cgacaccagc 240

ggctccacca actacaaccc cagcctgaaa tcgagggtta caatctctgt ggacacaagc 300

aagaatcagt tctccctgaa gctgagcagc gttactgccg ccgacacagc tgtgtactat 360

tgcgccagac tgggccagga aagcgctacc taccttggca tggatgtgtg ggggcagggc 420

accaccgtta ctgttagctc tggctcaaca agcggcagcg gcaagcctgg ctcaggagaa 480

ggaagcacaa agggcgacat tgtaatgact cagagccccg acagcctggc cgttagctta 540

ggcgaaaggg ctacaatcaa ttgcaagagc agccagagcg ttctgtacag cagcaacaac 600

aagaactacc tcgcatggta tcaacagaag ccaggccagc ctcccaagct gctcatctac 660

tgggcttcca ccagagagag cggggttccc gatagattct ccggctccgg ttctggaaca 720

gatttcacgc tcacaatcag cagcttacag gccgaggatg tggctgtcta ctattgtcag 780

cagttgtaca cctacccctt cacattcggc ggaggcacca aggttgagat caaggctgct 840

gcattggata atgaaaaatc gaacggcaca atcattcatg tgaagggcaa acacctgtgt 900

cccagcccct tgttcccagg acctagcaag cctttttggg ttctcgtggt ggtgggcggc 960

gttctggctt gctactctct acttgtaact gtcgcattta ttatattctg ggttagattc 1020

agcgttgtga agagaggccg gaagaagctg ctgtacatct tcaagcagcc cttcatgaga 1080

cctgtgcaga ccacacagga ggaagacggc tgcagctgta gattccccga ggaagaggag 1140

ggcggctgtg agctgagagt taagttcagc aggagcgccg acgcccctgc ctaccagcaa 1200

ggacagaatc aactgtacaa cgagctgaac ctgggcagac gggaggaata cgatgtgctg 1260

gacaagagga gaggcagaga ccccgagatg ggcggcaaac ctagaagaaa gaacccccag 1320

gagggcctgt ataacgagct ccagaaggac aagatggccg aggcctacag cgagatcggc 1380

atgaagggcg aaagaagaag aggcaagggc cacgacggcc tctaccaggg cttaagcaca 1440

gctacaaagg acacctacga cgccctgcac atgcaggccc tgccccctag atgattaatt 1500

aaatcgat 1508

<210> 291

<211> 3057

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 291

atgctgctgc tggtgacatc tctgctgctt tgcgagctgc cccaccctgc cttcctgctt 60

atccccgaca ttcagatgac ccagaccacc agcagcctga gcgccagctt aggagataga 120

gttaccatca gctgcagagc cagccaggac atcagcaaat acctgaactg gtatcagcag 180

aagcccgacg gcactgtgaa actgcttatt taccacacct ccagactgca cagcggcgtt 240

cccagcagat tctctggcag cggatctgga accgactaca gcctcaccat ctccaacctg 300

gagcaggagg acatcgccac ctacttctgc cagcagggca acacactgcc ctacaccttc 360

ggaggaggaa ccaagctgga gatcaccggg ggaggaggct ctggaggcgg cggatcagga 420

ggagggggat ctgaggttaa gctgcaggag agcggccctg gcctggtggc tcctagccaa 480

tctttatctg tgacctgcac tgtgtccggc gttagcctgc ccgattatgg cgtttcctgg 540

atcagacagc cccccagaaa gggcctggaa tggctgggcg ttatctgggg cagcgagacc 600

acatactaca acagcgccct gaagagcaga cttacgatta tcaaggacaa cagcaagagc 660

caggttttcc tgaagatgaa cagcctgcag accgacgaca ccgccatcta ctactgcgct 720

aagcactact actacggcgg cagctacgcc atggactact ggggccaggg aacaagcgtt 780

accgttagca gcgctgctgc actggacaac gagaagagca acggcaccat catccacgtt 840

aagggcaagc acctgtgccc cagccctctg ttccctggac cttctaagcc tttctgggtt 900

ctggtggtgg tcggcggcgt tttagcctgt tacagccttc tggtgactgt ggccttcatc 960

atcttttggg ttagaagcaa gagaagcaga ctgctccaca gcgactacat gaacatgacc 1020

cccagacggc ctggccccac cagaaagcat taccagccct acgctcctcc cagagacttc 1080

gccgcctaca ggagcagagt taaattcagc agatccgccg atgcccccgc ttaccaacag 1140

ggacaaaacc agctgtacaa tgagctcaac ctggggagaa gagaagaata cgacgttctg 1200

gataagagaa ggggcagaga tcccgaaatg gggggcaagc ccagacgcaa gaaccctcag 1260

gaggggcttt acaacgaact gcagaaggat aagatggctg aggcttactc ggagattggg 1320

atgaaggggg agagaaggcg gggcaaggga cacgatggct tataccaggg gctgagcacc 1380

gccaccaagg acacatacga cgctcttcat atgcaggctc tgcccccaag aagggctaag 1440

agatctggct ctggcgaggg cagaggcagc ttgcttacat gtggcgatgt ggaggagaac 1500

cccgggccca tggctcttcc tgtgacagct cttctgctgc ccctggccct gcttctgcat 1560

gctgctagac ctcagcttca gctccaagag agcggacctg gcttagtgaa gcccagcgaa 1620

accctgtccc tcacctgcac cgtttctggc ggaagcatca gcagctccag ctattactgg 1680

ggatggatca ggcagccccc tggcaagggt ttagaatgga tcggctcgat atattactcc 1740

ggcagcacct actataaccc cagcttgaag agccgggtta ccatttctgt ggacacatca 1800

aagaaccagt tcagcctgaa gctgagctct gtgactgccg ccgacacagc tgtgtactac 1860

tgtgccagag agacagacta ctccagcggc atgggctacg gcatggatgt gtggggacaa 1920

ggaaccaccg ttactgtgag cagcggttcc accagcggct caggcaagcc tggctcagga 1980

gaaggaagca ccaaggggga tatacagatg acacagagcc cctccagcct gtccgccagc 2040

gttggcgatc gtgtaacgat cacctgccgg gcctctcaga gcatcaactc ctacctcaat 2100

tggtatcaac agaagccagg caaggccccc aaattactca tctacgccgc cagcagctta 2160

cagagcgggg ttccctctag attctccggc tccggttctg gaacagattt caccctcact 2220

atctccagct tgcagcccga ggatttcgcc acttattact gtcagcagag cctggccgac 2280

cccttcacat tcggcggagg cacaaaggtt gagatcaagg cagctgcttt cgtgcctgtg 2340

ttcctgcctg ctaagcccac caccactcct gctccaagac ctcctacccc cgctcctaca 2400

atcgccagcc aacctctgag cctgagaccg gaggcatgca gacctgcggc agggggagca 2460

gttcacacaa gaggcttgga cttcgcttgc gacatctaca tctgggcccc tctggccggc 2520

acatgcggag ttcttcttct tagcctggtg atcaccctgt actgcaacca cagaaacaga 2580

ttcagcgttg tgaagagagg ccggaagaag ctgctgtaca tcttcaagca gcccttcatg 2640

agacctgtgc agaccacaca ggaggaagac ggctgcagct gtagattccc cgaggaagag 2700

gagggcggct gtgagctgag agttaagttc agcaggagcg ccgacgcccc tgcctaccag 2760

caaggacaga atcaactgta caacgagctg aacctgggca gacgggagga atacgatgtg 2820

ctggacaaga ggagaggcag agaccccgag atgggcggca aacctagaag aaagaacccc 2880

caggagggcc tgtataacga gctccagaag gacaagatgg ccgaggccta cagcgagatc 2940

ggcatgaagg gcgaaagaag aagaggcaag ggccacgacg gcctctacca gggcttaagc 3000

acagctacaa aggacaccta cgacgccctg cacatgcagg ccctgccccc tagatga 3057

<210> 292

<211> 1018

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 292

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

145 150 155 160

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

165 170 175

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

180 185 190

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

195 200 205

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

210 215 220

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

245 250 255

Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Leu Asp Asn Glu Lys

260 265 270

Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser

275 280 285

Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val

290 295 300

Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile

305 310 315 320

Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr

325 330 335

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

340 345 350

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys

355 360 365

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

370 375 380

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

385 390 395 400

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

405 410 415

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

420 425 430

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

435 440 445

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

450 455 460

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Lys

465 470 475 480

Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

485 490 495

Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu

500 505 510

Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gln Leu Gln Leu

515 520 525

Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu

530 535 540

Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp

545 550 555 560

Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser

565 570 575

Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg

580 585 590

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

595 600 605

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu

610 615 620

Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp Val Trp Gly Gln

625 630 635 640

Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys

645 650 655

Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr Gln

660 665 670

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

675 680 685

Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn Trp Tyr Gln Gln

690 695 700

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu

705 710 715 720

Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

725 730 735

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

740 745 750

Tyr Cys Gln Gln Ser Leu Ala Asp Pro Phe Thr Phe Gly Gly Gly Thr

755 760 765

Lys Val Glu Ile Lys Ala Ala Ala Phe Val Pro Val Phe Leu Pro Ala

770 775 780

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

785 790 795 800

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

805 810 815

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

820 825 830

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

835 840 845

Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Phe Ser Val Val

850 855 860

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

865 870 875 880

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

885 890 895

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

900 905 910

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

915 920 925

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

930 935 940

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

945 950 955 960

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

965 970 975

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

980 985 990

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

995 1000 1005

Ala Leu His Met Gln Ala Leu Pro Pro Arg

1010 1015

<210> 293

<211> 2202

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polynucleotide

<400> 293

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgccgggc aagtcagagc attaacagct atttaaattg gtatcagcag 180

aaaccaggga aagcccctaa gctcctgatc tatgctgcat ccagtttgca aagtggggtc 240

ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 300

caacctgaag attttgcaac ttactactgc cagcaaagcc tcgccgaccc tttcactttt 360

ggcggaggga ccaaggttga gatcaaaggg gggggtggaa gtgggaagcc tggcagcggc 420

gagggcggca gtcagctgca gctgcaggag tcgggcccag gactggtgaa gccttcggag 480

accctgtccc tcacctgcac tgtctctggt ggctccatca gcagtagtag ttactactgg 540

ggctggatcc gccagccccc agggaagggg ctggagtgga ttgggagtat ctattatagt 600

gggagcacct actacaaccc gtccctcaag agtcgagtca ccatatccgt agacacgtcc 660

aagaaccagt tctccctgaa gctgagttct gtgaccgccg cagacacggc ggtgtactac 720

tgcgccagag agactgacta cagcagcgga atgggatacg gaatggacgt atggggccag 780

ggaacaactg tcaccgtctc ctcaggcggt ggcggcagtg ggaagcctgg cagcgatatt 840

caaatgaccc agtccccgtc ctccctgagt gcctccgtcg gtgaccgtgt tacgattacc 900

tgccgtgcga gccaagacat ctctaaatac ctgaactggt atcagcaaaa accggatcag 960

gcaccgaaac tgctgatcaa acatacctca cgtctgcact cgggtgtgcc gagccgcttt 1020

agtggttccg gctcaggtac cgattacacc ctgacgatca gctctctgca gccggaagac 1080

tttgccacgt attactgcca gcaaggtaat accctgccgt atacgttcgg ccaaggtacc 1140

aaactggaaa tcaaaggggg gggtggaagt gggggcggtg gcagcggcgg tggcggcagt 1200

gaagtgcagc tggttgaaag cggtggtggt ctggttcaac cgggtcgttc cctgcgtctg 1260

tcatgtacgg cgagtggtgt ctccctgccg gactatggcg tgtcctggat tcgtcagccg 1320

ccgggtaaag gcctggaatg gattggtgtc atctggggca gtgaaaccac gtattacaac 1380

tcggccctga aaagccgttt caccatctct cgcgataaca gtaaaaatac gctgtacctg 1440

cagatgaata gcctgcgcgc ggaagacacc gccgtttact actgcgcaaa acattactac 1500

tacggtggca gctatgctat ggattactgg ggtcaaggca cgctggtcac cgtttcgtca 1560

gccgctgccc tagacaatga gaagagcaat ggaaccatta tccatgtgaa agggaaacac 1620

ctttgtccaa gtcccctatt tcccggacct tctaagccct tttgggtgct ggtggtggtt 1680

gggggagtcc tggcttgcta tagcttgcta gtaacagtgg cctttattat tttctgggtg 1740

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1800

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1860

tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1920

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1980

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 2040

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 2100

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 2160

acctacgacg cccttcacat gcaggccctg ccccctcgat ga 2202

<210> 294

<211> 733

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 294

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Ser Ile Asn Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser Leu Ala Asp Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Gly Gly Gly Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Gly Ser

130 135 140

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

145 150 155 160

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

165 170 175

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

180 185 190

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

195 200 205

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

210 215 220

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

225 230 235 240

Cys Ala Arg Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp

245 250 255

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

260 265 270

Ser Gly Lys Pro Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

275 280 285

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

290 295 300

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gln

305 310 315 320

Ala Pro Lys Leu Leu Ile Lys His Thr Ser Arg Leu His Ser Gly Val

325 330 335

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr

340 345 350

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

355 360 365

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

370 375 380

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

405 410 415

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Ser Leu Pro Asp Tyr

420 425 430

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

435 440 445

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

450 455 460

Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

465 470 475 480

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

485 490 495

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

500 505 510

Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Leu Asp Asn Glu Lys

515 520 525

Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser

530 535 540

Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val

545 550 555 560

Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile

565 570 575

Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr

580 585 590

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

595 600 605

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys

610 615 620

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

625 630 635 640

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

645 650 655

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

660 665 670

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

675 680 685

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

690 695 700

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

705 710 715 720

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

725 730

<210> 295

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 295

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 296

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 296

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Ala Asp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 297

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 297

Gly Gly Gly Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Gly Ser

1 5 10 15

<210> 298

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 298

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Thr Asp Tyr Ser Ser Gly Met Gly Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 299

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 299

Gly Gly Gly Gly Ser Gly Lys Pro Gly Ser

1 5 10

<210> 300

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 300

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gln Ala Pro Lys Leu Leu Ile

35 40 45

Lys His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 301

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 301

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 302

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 302

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 303

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 303

Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys

1 5 10 15

His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro

20 25 30

<210> 304

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 304

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 305

<211> 41

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 305

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 306

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<400> 306

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 307

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 307

Gly Gly Gly Gly Ser

1 5

<210> 308

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 308

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 309

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 309

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 310

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 310

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 311

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<400> 311

Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser

1 5 10 15

Pro Ser

<210> 312

<211> 50

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<220>

<221> MISC_FEATURE

<222> (1)..(50)

<223> the sequence can cover 1-10 "Pro Ala Pro Ala Pro"

Repeating unit

<400> 312

Pro Ala Pro Ala Pro Pro Ala Pro Ala Pro Pro Ala Pro Ala Pro Pro

1 5 10 15

Ala Pro Ala Pro Pro Ala Pro Ala Pro Pro Ala Pro Ala Pro Pro Ala

20 25 30

Pro Ala Pro Pro Ala Pro Ala Pro Pro Ala Pro Ala Pro Pro Ala Pro

35 40 45

Ala Pro

50

<210> 313

<211> 50

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Polypeptides

<220>

<221> MISC_FEATURE

<222> (1)..(50)

<223> the sequence can cover 1-10 "Glu Ala Ala Ala Lys"

Repeating unit

<400> 313

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala

20 25 30

Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala

35 40 45

Ala Lys

50

<210> 314

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences Synthesis

Peptides

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Val or Ile

<220>

<221> MOD_RES

<222> (4)..(4)

<223> any amino acid

<400> 314

Asp Xaa Glu Xaa Asn Pro Gly Pro

1 5

Claims

1. An antigen binding system, antibody or antigen binding fragment thereof comprising an anti-CD 20 binding motif, wherein the anti-CD 20 binding motif comprises the sequence of three heavy chain complementarity determining regions (HCDRs) of any one of the Heavy Chain Variable Regions (HCVRs) selected from the group consisting of seq id nos: 1, 23, 45, 67, 89, 111, 133, 155, 177 and 199, and the sequence of the three light chain cdrs (lcdrs) of the Light Chain Variable Region (LCVR) selected from the group consisting of SEQ ID NOs: 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210.

2. The antigen binding system, antibody or antigen binding fragment thereof of claim 1, wherein the anti-CD 20 binding motif comprises a first domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and a second domain comprising three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein

(i) The HCDR1 has the sequences shown in SEQ ID NO:3-5, 25-27, 47-49, 69-71, 91-93, 113-; and any one of 201-203;

(ii) the HCDR2 has the sequences shown in SEQ ID NOs 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184; and 204-206;

(iii) the HCDR3 has the structure shown in SEQ ID NO 9-11, 31-33, 53-55, 75-77, 97-99, 119-121, 141-143, 163-165, 185-187; and 207-209;

(iv) the LCDR1 has the sequences shown in SEQ ID NO 14-16, 36-38, 58-60, 80-82, 102-104, 124-126, 146-148, 168-170, 190-192; and 212-214;

(v) the LCDR2 has the sequences shown in SEQ ID NO 17-19, 39-41, 61-63, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and is

(vi) The LCDR3 has the sequences shown in SEQ ID NO:20-22, 42-44, 64-66, 86-88, 108-; and any of 218 and 220.

3. The antigen binding system, antibody or antigen binding fragment thereof of claim 1 or 2, wherein the HCDR comprises:

(i) HCDR1 according to any one of SEQ ID NOs 3-5; HCDR2 according to any one of SEQ ID NOs 6-8; HCDR3 according to any one of SEQ ID NOs 9-11;

(ii) HCDR1 according to any one of SEQ ID NOs 25-27; HCDR2 according to any one of SEQ ID NOs 28-30; HCDR3 according to any one of SEQ ID NOs 31-33;

(iii) HCDR1 according to any one of SEQ ID NOs 47-49; HCDR2 according to any one of SEQ ID NOs 50-52; HCDR3 according to any one of SEQ ID NOs 53-55;

(iv) HCDR1 according to any one of SEQ ID NOs 69-71; HCDR2 according to any one of SEQ ID NOs 72-74; HCDR3 according to any one of SEQ ID NOs 75-77;

(v) HCDR1 according to any one of SEQ ID NOs 91-93; HCDR2 according to any one of SEQ ID NOs 94-96; HCDR3 according to any one of SEQ ID NOs 97-99;

(vi) HCDR1 according to any one of SEQ ID NO 113-115; HCDR2 according to any one of SEQ ID NO: 116-118; HCDR3 according to any one of SEQ ID NO 119-121;

(vii) HCDR1 according to any one of SEQ ID NO 135-137; HCDR2 according to any one of SEQ ID NO 138-140; HCDR3 according to any one of SEQ ID NO 141-143;

(viii) HCDR1 according to any one of SEQ ID NO 157-159; HCDR2 according to any one of SEQ ID NOs 160-162; HCDR3 according to any one of SEQ ID NO 163-165;

(ix) HCDR1 according to any one of SEQ ID NO 179-181; HCDR2 according to any one of SEQ ID NO 182-184; HCDR3 according to any one of SEQ ID NO 185-187; or

(x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO 204-206; HCDR3 according to any one of SEQ ID NO 207-209; and is

The LCDR comprises:

(i) LCDR1 according to any one of SEQ ID NOs 14-16; LCDR2 according to any one of SEQ ID NOs 17-19; LCDR3 according to any one of SEQ ID NOs 20-22;

(ii) LCDR1 according to any one of SEQ ID NOs 36-38; LCDR2 according to any one of SEQ ID NOs 39-41; LCDR3 according to any one of SEQ ID NOS 42-44;

(iii) LCDR1 according to any one of SEQ ID NOs 58-60; LCDR2 according to any one of SEQ ID NOs 61-63; LCDR3 according to any one of SEQ ID NOs 64-66;

(iv) LCDR1 according to any one of SEQ ID NOs 80-82; LCDR2 according to any one of SEQ ID NOs 83-85; LCDR3 according to any one of SEQ ID NOs 86-88;

(v) LCDR1 according to any one of SEQ ID NO 102-104; LCDR2 according to any one of SEQ ID NO 105-107; LCDR3 according to any one of SEQ ID NO 108-110;

(vi) LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO 127-129; LCDR3 according to any one of SEQ ID NO: 130-132;

(vii) LCDR1 according to any one of SEQ ID NO 146-148; LCDR2 according to any one of SEQ ID NO 149-151; LCDR3 according to any one of SEQ ID NO 152-154;

(viii) LCDR1 according to any one of SEQ ID NO 168-170; LCDR2 according to any one of SEQ ID NO 171-173; LCDR3 according to any one of SEQ ID NO: 174-176;

(ix) LCDR1 according to any one of SEQ ID NO 190-192; LCDR2 according to any one of SEQ ID NO 193-195; LCDR3 according to any one of SEQ ID NO 196-198; or

(x) LCDR1 according to any one of SEQ ID NO 212-214; LCDR2 according to any of SEQ ID NO:215 and 217; LCDR3 according to any one of SEQ ID NO 218-220.

4. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-3, wherein the antigen binding system, antibody or antigen binding fragment thereof comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), wherein:

The HCDR and LCDR comprise:

(i) HCDR1 according to any one of SEQ ID NOs 3-5; HCDR2 according to any one of SEQ ID NOs 6-8; HCDR3 according to any one of SEQ ID NOs 9-11; LCDR1 according to any one of SEQ ID NOs 14-16; LCDR2 according to any one of SEQ ID NOs 17-19; LCDR3 according to any one of SEQ ID NOs 20-22;

(ii) HCDR1 according to any one of SEQ ID NOs 25-27; HCDR2 according to any one of SEQ ID NOs 28-30; HCDR3 according to any one of SEQ ID NOs 31-33; LCDR1 according to any one of SEQ ID NOs 36-38; LCDR2 according to any one of SEQ ID NOs 39-41; LCDR3 according to any one of SEQ ID NOS 42-44;

(iii) HCDR1 according to any one of SEQ ID NOs 47-49; HCDR2 according to any one of SEQ ID NOs 50-52; HCDR3 according to any one of SEQ ID NOs 53-55; LCDR1 according to any one of SEQ ID NOs 58-60; LCDR2 according to any one of SEQ ID NOs 61-63; LCDR3 according to any one of SEQ ID NOs 64-66;

(iv) HCDR1 according to any one of SEQ ID NOs 69-71; HCDR2 according to any one of SEQ ID NOs 72-74; HCDR3 according to any one of SEQ ID NOs 75-77; LCDR1 according to any one of SEQ ID NOs 80-82; LCDR2 according to any one of SEQ ID NOs 83-85; LCDR3 according to any one of SEQ ID NOs 86-88;

(v) HCDR1 according to any one of SEQ ID NOs 91-93; HCDR2 according to any one of SEQ ID NOs 94-96; HCDR3 according to any one of SEQ ID NOs 97-99; LCDR1 according to any one of SEQ ID NO 102-104; LCDR2 according to any one of SEQ ID NO 105-107; LCDR3 according to any one of SEQ ID NO 108-110;

(vi) HCDR1 according to any one of SEQ ID NO 113-115; HCDR2 according to any one of SEQ ID NO: 116-118; HCDR3 according to any one of SEQ ID NO 119-121; LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO 127-129; LCDR3 according to any one of SEQ ID NO: 130-132;

(vii) HCDR1 according to any one of SEQ ID NO 135-137; HCDR2 according to any one of SEQ ID NO 138-140; HCDR3 according to any one of SEQ ID NO 141-143; LCDR1 according to any one of SEQ ID NO 146-148; LCDR2 according to any one of SEQ ID NO 149-151; LCDR3 according to any one of SEQ ID NO 152-154;

(viii) HCDR1 according to any one of SEQ ID NO 157-159; HCDR2 according to any one of SEQ ID NOs 160-162; HCDR3 according to any one of SEQ ID NO 163-165; LCDR1 according to any one of SEQ ID NO 168-170; LCDR2 according to any one of SEQ ID NO 171-173; LCDR3 according to any one of SEQ ID NO: 174-176;

(ix) HCDR1 according to any one of SEQ ID NO 179-181; HCDR2 according to any one of SEQ ID NO 182-184; HCDR3 according to any one of SEQ ID NO 185-187; LCDR1 according to any one of SEQ ID NO 190-192; LCDR2 according to any one of SEQ ID NO 193-195; LCDR3 according to any one of SEQ ID NO 196-198; or

(x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO 204-206; HCDR3 according to any one of SEQ ID NO 207-209; LCDR1 according to any one of SEQ ID NO 212-214; LCDR2 according to any of SEQ ID NO:215 and 217; LCDR3 according to any one of SEQ ID NO 218-220.

5. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-4, wherein the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs, wherein:

(i) the heavy chain variable domain is at least 80% identical to SEQ ID NO 1, 23, 45, 67, 89, 111, 133, 155, 177 or 199; and is

(ii) The light chain variable domain is at least 80% identical to SEQ ID NO 12, 34, 56, 78, 100, 122, 144, 166, 188 or 210.

6. The antigen binding system, antibody or antigen binding fragment thereof of claim 5, wherein the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs, wherein:

(i) the heavy chain variable domain is at least 80% identical to SEQ ID NO 1 and the light chain variable domain is at least 80% identical to SEQ ID NO 12;

(ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 23 and the light chain variable domain is at least 80% identical to SEQ ID NO 34;

(iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 45 and the light chain variable domain is at least 80% identical to SEQ ID NO 56;

(iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO 67 and the light chain variable domain is at least 80% identical to SEQ ID NO 78;

(v) the heavy chain variable domain is at least 80% identical to SEQ ID NO. 89 and the light chain variable domain is at least 80% identical to SEQ ID NO. 100;

(vi) The heavy chain variable domain is at least 80% identical to SEQ ID NO 111 and the light chain variable domain is at least 80% identical to SEQ ID NO 122;

(vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO 133 and the light chain variable domain is at least 80% identical to SEQ ID NO 144;

(viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:155 and the light chain variable domain is at least 80% identical to SEQ ID NO: 166;

(ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO:177 and the light chain variable domain is at least 80% identical to SEQ ID NO: 188; or

(x) The heavy chain variable domain is at least 80% identical to SEQ ID NO 199 and the light chain variable domain is at least 80% identical to SEQ ID NO 210.

7. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-6, wherein the three HCDRs and the three LCDRs are comprised by a single polypeptide.

8. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-6, wherein the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide.

9. The antigen binding system, antibody or antigen binding fragment thereof of claim 8, wherein said first polypeptide is an antibody heavy chain and said second polypeptide is an antibody light chain.

10. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-9, wherein the antigen binding system, antibody or antigen binding fragment thereof further comprises:

(i) a binding motif that specifically binds to an antigen selected from the group consisting of: 5T4, alpha-fetoprotein, B-cell maturation antigen (BCMA), B-cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, C-Met, CSPG4, IgM, C-type lectin-like molecule 1(CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT 2, folate binding protein, GD2, HER2-HER 2 combination, HER2/Neu, HERV-2-1 envelope glycoprotein gp 2, HIV-1 envelope glycoprotein gpl2, IL-rall 2, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p 2, mutated ras, VEGFR 72, VEGFR-specific receptor (ephfr 2), ephha 2), and ephha receptor 2 (ephha 2); and/or

(ii) A binding motif that specifically binds to a B cell characteristic antigen, optionally wherein the B cell characteristic antigen is not CD19 or CD 20.

11. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-9, wherein said antigen binding system, antibody or antigen binding fragment thereof further comprises an anti-CD 19 binding motif.

12. The antigen binding system, antibody or antigen binding fragment thereof of claim 11, wherein the anti-CD 19 binding motif comprises a first domain comprising three HCDRs and a second domain comprising three LCDRs, wherein:

the three HCDRs of the anti-CD 19 binding motif comprise HCDR1, HCDR2, and HCDR 3;

the three LCDRs of the anti-CD 19 binding motif comprise LCDR1, LCDR2, and LCDR 3; and is

The HCDR and LCDR of the anti-CD 19 binding motif comprise HCDR1 according to any one of SEQ ID NO: 223-225; HCDR2 according to any one of SEQ ID NO 226-228; HCDR3 according to any one of SEQ ID NO 229-231; LCDR1 according to any one of SEQ ID NO 234-236; LCDR2 according to any one of SEQ ID NO 237 and 239; LCDR3 according to any one of SEQ ID NO 240-242.

13. The antigen binding system, antibody or antigen binding fragment thereof of claim 12, wherein the anti-CD 19 binding motif comprises a first heavy chain variable domain comprising the three HCDRs of the anti-CD 19 binding motif and a light chain variable domain comprising the three LCDRs of the anti-CD 19 binding motif, wherein the heavy chain variable domain of the anti-CD 19 binding motif is at least 80% identical to SEQ ID No. 221 and the light chain variable domain of the anti-CD 19 binding motif is at least 80% identical to SEQ ID No. 232.

14. The antigen binding system, antibody or antigen binding fragment thereof of claim 12 or claim 13, wherein the three HCDRs of the anti-CD 19 binding motif and the three LCDRs of the anti-CD 19 binding motif are comprised by a single polypeptide.

15. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 12-14, wherein the three HCDRs of the anti-CD 20 binding motif, the three LCDRs of the anti-CD 20 binding motif, the three HCDRs of the anti-CD 19 binding motif, and the three LCDRs of the anti-CD 19 binding motif together are comprised by a single polypeptide.

16. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-15, wherein said antigen binding system, antibody or antigen binding fragment thereof is or is comprised by a chimeric antigen receptor.

17. The antigen binding system, antibody or antigen binding fragment thereof of claim 16, wherein said chimeric antigen receptor comprises a transmembrane domain that is a transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.

18. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 11-14, wherein (i) the three HCDRs of the anti-CD 20 binding motif and the three LCDRs of the anti-CD 20 binding motif are present in a first polypeptide, and (ii) the three HCDRs of the anti-CD 19 binding motif and the three LCDRs of the anti-CD 19 binding motif together are comprised by a second, different polypeptide.

19. The antigen binding system, antibody or antigen binding fragment thereof of claim 18, wherein said first polypeptide is or is comprised by a first chimeric antigen receptor.

20. The antigen binding system, antibody or antigen binding fragment thereof of claim 18 or claim 19, wherein the second polypeptide is or is comprised by a second chimeric antigen receptor.

21. A nucleic acid encoding at least one polypeptide of any one of claims 1-20.

22. A vector comprising the nucleic acid of claim 21.

23. A method of producing an engineered cell, the method comprising transfecting or transducing a cell with a nucleic acid according to claim 21 or a vector according to claim 22.

24. A cell encoding or expressing the antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-20, optionally wherein the cell is an immune cell, optionally wherein the cell is a T cell.

25. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising the antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1-20.

26. A method of inducing an immune response in a subject or immunizing a subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising the antigen binding system, antibody or antigen binding fragment of any one of claims 1-20.

27. The method of claim 25 or 26, wherein the cell is a CAR-T cell.

28. The method of any one of claims 25-27, wherein the cancer is Acute Lymphoblastic Leukemia (ALL) (including non-T cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, burkitt's lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), Follicular Lymphoma (FL), hairy cell leukemia, hodgkin's disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, Monoclonal Gammopathy of Unknown Significance (MGUS), multiple myeloma, myelodysplasia, and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), a plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmacytoma-like dendritic cell neoplasms, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsutsi disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small-or large-cell follicular lymphoma, splenic marginal zone lymphoma (SMZLL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma or Wallace macroglobulinemia, Mantle Cell Lymphoma (MCL), Transformed Follicular Lymphoma (TFL), Primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, hairy cell lymphoma/leukemia, or a combination thereof.

29. The method of any one of claims 25-28, wherein the cell therapy is allogeneic or autologous cell therapy.