HK40061606B - Chimeric antigen receptors and car-t cells and methods of use - Google Patents

Description

Chimeric antigens and T-cell receptors and their application methods

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 62/778,893, filed December 12, 2018, which is incorporated herein by reference in its entirety.

Background of the Invention

Human cancer, by its very nature, consists of normal cells that have undergone genetic or epigenetic transformation into abnormal cancer cells. At this point, cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. These abnormal 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 them.

Current T-cell therapies rely on enriched or modified human T cells to target and kill cancer cells in patients. To enhance 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. These include chimeric antigen receptors (CARs) and engineered T-cell receptors (TCRs), which contain binding domains capable of interacting with specific tumor antigens, allowing T cells to target and kill cancer cells expressing those specific tumor antigens. There is a need for CARs and TCRs for targeting and killing cancer cells.

Invention Overview

In at least a first aspect, this disclosure includes an antigen-binding system comprising an anti-CD20 binding motif, an antibody, or an antigen-binding fragment thereof, wherein the anti-CD20 binding motif comprises sequences of three heavy chain complementarity-determining regions (HCDRs) of any heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO: 1, 23, 45, 67, 89, 111, 133, 155, 177, and 199 and sequences of three light chain CDRs (LCDRs) of any light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 12, 34, 56, 78, 100, 122, 144, 166, 188, and 210. In some embodiments, the anti-CD20 binding motif comprises a first domain containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and a second domain containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein (i) HCDR1 has according to SEQ ID NO: 3-5, 25-27, 47-49, 69-71, 91-93, 113-115, 135-137, 157-159, 179-1 81; and any one of 201-203; (ii) HCDR2 has a sequence according to any one of SEQ ID NO: 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184 and 204-206; (iii) HCDR3 has a sequence according to any one of SEQ ID NO: 9-11, 31-33, 53-55, 75-77, 97-99, 119- (iv) LCDR1 has a sequence according to any one of 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) LCDR2 has a sequence according to SEQ ID NO: 17-19, 39 -41, 61-63, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and any one of 215-217; and (vi)LCDR3 has a sequence according to SEQ ID NO: 20-22, 42-44, 64-66, 86-88, 108-110, 130-132, 152-154, 174-176, 196-198 and 218-220. In some implementations, the HCDR comprises: (i) HCDR1 according to any one of SEQ ID NO: 3-5; HCDR2 according to any one of SEQ ID NO: 6-8; HCDR3 according to any one of SEQ ID NO: 9-11; (ii) HCDR1 according to any one of SEQ ID NO: 25-27; HCDR2 according to any one of SEQ ID NO: 28-30; HCDR3 according to any one of SEQ ID NO: 31-33; (iii) HCDR1 according to any one of SEQ ID NO: 47-49; HCDR3 according to any one of SEQ ID NO: 38-39; HCDR2 according to any one of SEQ ID NO: 38-39; HCDR3 ... HCDR2 of any one of SEQ ID NO: 50-52; HCDR3 of any one of SEQ ID NO: 53-55; (iv) HCDR1 of any one of SEQ ID NO: 69-71; HCDR2 of any one of SEQ ID NO: 72-74; HCDR3 of any one of SEQ ID NO: 75-77; (v) HCDR1 of any one of SEQ ID NO: 91-93; HCDR2 of any one of SEQ ID NO: 94-96; HCDR3 of any one of SEQ ID NO: 97-99; (vi) HCDR3 of any one of SEQ ID NO: 50-52; HCDR1 of any one of SEQ ID NO: 113-115; HCDR2 of any one of SEQ ID NO: 116-118; HCDR3 of any one of SEQ ID NO: 119-121; (vii) HCDR1 of any one of SEQ ID NO: 135-137; HCDR2 of any one of SEQ ID NO: 138-140; HCDR3 of any one of SEQ ID NO: 141-143; (viii) HCDR1 of any one of SEQ ID NO: 157-159; HCDR3 of any one of SEQ ID NO: 160-165. HCDR2 of any one of SEQ ID NO: 163-165; (ix) HCDR1 of any one of SEQ ID NO: 179-181; HCDR2 of any one of SEQ ID NO: 182-184; HCDR3 of any one of SEQ ID NO: 185-187; or (x) HCDR1 of any one of SEQ ID NO: 201-203; HCDR2 of any one of SEQ ID NO: 204-206; HCDR3 of any one of SEQ ID NO: 207-209; and The LCDR comprises: (i) LCDR1 according to any one of SEQ ID NO: 14-16; LCDR2 according to any one of SEQ ID NO: 17-19; LCDR3 according to any one of SEQ ID NO: 20-22; (ii) LCDR1 according to any one of SEQ ID NO: 36-38; LCDR2 according to any one of SEQ ID NO: 39-41; LCDR3 according to any one of SEQ ID NO: 42-44; (iii) LCDR1 according to any one of SEQ ID NO: 58-60; LCDR3 according to any one of SEQ ID NO: 61- LCDR2 according to any one of SEQ ID NO: 63; LCDR3 according to any one of SEQ ID NO: 64-66; (iv) LCDR1 according to any one of SEQ ID NO: 80-82; LCDR2 according to any one of SEQ ID NO: 83-85; LCDR3 according to any one of SEQ ID NO: 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) according to SEQ ID NO: 63-64-65-66-66-66-67-68-69-63-64-65-66-66-63-64-65-66-66-67-68-69-66-63-64-65-66-66-67-68-69-66-64-65-66-66-67-68-69-66-64-65-66-66-67-68-69-66-69-66-67-69-68-69 ... 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; LCDR3 according to any one of SEQ ID NO: 171-16 ...68-170; LCDR3 according to any one of SEQ ID NO: 171-168; LCDR2 according to any one of SEQ ID NO: 127-129; LCDR3 according to any one of SEQ ID NO: 130-132; 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; LCDR1 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 1 LCDR2 of any one of SEQ ID NO: 174-176; (ix) LCDR1 of any one of SEQ ID NO: 190-192; LCDR2 of any one of SEQ ID NO: 193-195; LCDR3 of any one of SEQ ID NO: 196-198; or (x) LCDR1 of any one of SEQ ID NO: 212-214; LCDR2 of any one of SEQ ID NO: 215-217; LCDR3 of any one of SEQ ID NO: 218-220.

In various embodiments, the antigen-binding system, antibody, or antigen-binding fragment thereof of this disclosure includes 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 HCDRs and LCDRs comprise: (i) HCDR1 according to any one of SEQ ID NO: 3-5; HCDR2 according to any one of SEQ ID NO: 6-8; HCDR3 according to any one of SEQ ID NO: 9-11; LCDR1 according to any one of SEQ ID NO: 14-16; LCDR2 according to any one of SEQ ID NO: 17-19; and so on. (ii) LCDR3 according to any one of SEQ ID NO: 20-22; (ii) HCDR1 according to any one of SEQ ID NO: 25-27; HCDR2 according to any one of SEQ ID NO: 28-30; HCDR3 according to any one of SEQ ID NO: 31-33; LCDR1 according to any one of SEQ ID NO: 36-38; LCDR2 according to any one of SEQ ID NO: 39-41; LCDR3 according to any one of SEQ ID NO: 42-44; (iii) HCDR1 according to any one of SEQ ID NO: 47-49; according to SE HCDR2 according to any one of SEQ ID NO: 50-52; HCDR3 according to any one of SEQ ID NO: 53-55; LCDR1 according to any one of SEQ ID NO: 58-60; LCDR2 according to any one of SEQ ID NO: 61-63; LCDR3 according to any one of SEQ ID NO: 64-66; (iv) HCDR1 according to any one of SEQ ID NO: 69-71; HCDR2 according to any one of SEQ ID NO: 72-74; HCDR3 according to any one of SEQ ID NO: 75-77; according to SEQ ID NO: 50-52; HCDR2 according to any one of SEQ ID NO: 53-55; LCDR1 according to any one of SEQ ID NO: 58-60; LCDR2 according to any one of SEQ ID NO: 61-63; LCDR3 according to any one of SEQ ID NO: 64-66; (iv) HCDR1 according to any one of SEQ ID NO: 69-71; HCDR2 according to any one of SEQ ID NO: 72-74; HCDR3 according to any one of SEQ ID NO: 75-77; LCDR3 according to any one of SEQ ID NO: 58-60; LCDR2 according to any one of SEQ ID NO: 69-71; LCDR2 according to any one of SEQ ID NO: 72-74; LCDR3 according to any one of SEQ ID NO: 75-77; LCDR3 ... LCDR1 according to any one of SEQ ID NO: 80-82; LCDR2 according to any one of SEQ ID NO: 83-85; LCDR3 according to any one of SEQ ID NO: 86-88; (v) HCDR1 according to any one of SEQ ID NO: 91-93; HCDR2 according to any one of SEQ ID NO: 94-96; HCDR3 according to any one of SEQ ID NO: 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: 10 LCDR3 of any one of SEQ ID NO: 113-115; HCDR1 of any one of SEQ ID NO: 116-118; HCDR3 of any one of SEQ ID NO: 119-121; LCDR1 of any one of SEQ ID NO: 124-126; LCDR2 of any one of SEQ ID NO: 127-129; LCDR3 of any one of SEQ ID NO: 130-132; (vii) HCDR1 of any one of SEQ ID NO: 135-137; HCDR2 of any one of SEQ ID NO: 138-140; HCDR3 of any one of SEQ ID NO: 141-143; LCDR1 of any one of SEQ ID NO: 146-148; LCDR2 of any one of SEQ ID NO: 149-151; LCDR3 of any one of SEQ ID NO: 152-154; (viii) HCDR1 of any one of SEQ ID NO: 157-159; HCDR2 of any one of SEQ ID NO: 160-162; HCDR3 of any one of SEQ ID NO: 163-165 HCDR3 of any one of SEQ ID NO: 168-170; LCDR1 of any one of SEQ ID NO: 171-173; LCDR2 of any one of SEQ ID NO: 174-176; (ix) HCDR1 of any one of SEQ ID NO: 179-181; HCDR2 of any one of SEQ ID NO: 182-184; HCDR3 of any one of SEQ ID NO: 185-187; LCDR1 of any one of SEQ ID NO: 190-192; LCDR3 ... O: LCDR2 of any one of SEQ ID NO: 193-195; LCDR3 of any one of SEQ ID NO: 196-198; or (x) HCDR1 of any one of SEQ ID NO: 201-203; HCDR2 of any one of SEQ ID NO: 204-206; HCDR3 of any one of SEQ ID NO: 207-209; LCDR1 of any one of SEQ ID NO: 212-214; LCDR2 of any one of SEQ ID NO: 215-217; LCDR3 of any one of SEQ ID NO: 218-220. In some embodiments, the antigen-binding system, antibody, or antigen-binding fragment thereof includes 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 consistent with SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:67, SEQ ID NO:89, SEQ ID NO:111, SEQ ID NO:133, SEQ ID NO:15 ...3, SEQ ID NO:67, SEQ ID NO:89, SEQ ID NO:111, SEQ ID NO:133, SEQ ID NO:155, SEQ ID NO:13, SEQ ID NO:133, SEQ ID NO:155, SEQ ID NO:133, SEQ ID NO:133, SEQ ID NO:155, SEQ ID NO:133, SEQ ID NO:1 (ii) The light chain variable structural domain is at least 80% identical to SEQ ID NO:177 or SEQ ID NO:199; and (ii) the light chain variable structural domain is at least 80% identical to SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:56, SEQ ID NO:78, SEQ ID NO:100, SEQ ID NO:122, SEQ ID NO:144, SEQ ID NO:166, SEQ ID NO:188 or SEQ ID NO:210. In some embodiments, the antigen-binding system, antibody, or antigen-binding fragment thereof includes 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:89 and the light chain variable domain is at least 80% identical to SEQ ID NO:89. The variable structural domains are at least 80% identical to SEQ ID NO:100; (vi) the heavy chain variable structural domains are at least 80% identical to SEQ ID NO:111 and the light chain variable structural domains are at least 80% identical to SEQ ID NO:122; (vii) the heavy chain variable structural domains are at least 80% identical to SEQ ID NO:133 and the light chain variable structural domains are at least 80% identical to SEQ ID NO:144; (viii) the heavy chain variable structural domains are at least 80% identical to SEQ ID NO:144. (ix) The variable domains of the heavy chain are at least 80% identical to those of SEQ ID NO:155 and at least 80% identical to those of SEQ ID NO:166; or (x) The variable domains of the heavy chain are at least 80% identical to those of SEQ ID NO:177 and at least 80% identical to those of SEQ ID NO:188; or (x) The variable domains of the heavy chain are at least 80% identical to those of SEQ ID NO:199 and at least 80% identical to those of SEQ ID NO:210.

This disclosure includes some embodiments of three HCDRs and three LCDRs, wherein the three HCDRs and three LCDRs are comprised of a single polypeptide. In some embodiments of this disclosure, the three HCDRs are comprised of a first polypeptide and the three LCDRs are comprised of 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 binding 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, FLT3, folate-binding protein, GD2, GD3, HER1- HER2 combination, HER2-HER3 combination, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B cell activating factor receptor), and combinations thereof; and/or (ii) binding motifs binding to B cell characteristic antigens, optionally wherein the B cell characteristic antigen is not CD19 or CD20. In some embodiments, the antigen-binding system, antibody, or antigen-binding fragment thereof further comprises an anti-CD19 binding motif. In some embodiments, the anti-CD19 binding motif includes a first domain comprising three HCDRs and a second domain comprising three LCDRs, wherein: the three HCDRs of the anti-CD19 binding motif comprise HCDR1, HCDR2, and HCDR3; the three LCDRs of the anti-CD19 binding motif comprise LCDR1, LCDR2, and LCDR3; and the HCDRs and LCDRs of the anti-CD19 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: 237-239; and LCDR3 according to any one of SEQ ID NO: 240-242. In some embodiments, the anti-CD19 binding motif comprises a first heavy chain variable domain containing three HCDRs of the anti-CD19 binding motif and a light chain variable domain containing three LCDRs of the anti-CD19 binding motif, wherein the heavy chain variable domain of the anti-CD19 binding motif is at least 80% identical to SEQ ID NO:221, and the light chain variable domain of the anti-CD19 binding motif is at least 80% identical to SEQ ID NO:232. In some embodiments, the three HCDRs and three LCDRs of the anti-CD19 binding motif are contained in a single polypeptide. In some embodiments, the three HCDRs, three LCDRs, three HCDRs, and three LCDRs of the anti-CD20 binding motif are collectively contained in a single polypeptide.

In various embodiments, the antigen-binding system, antibody, or antigen-binding fragment thereof is a chimeric antigen receptor or is contained within a chimeric antigen receptor. In some embodiments, the antigen-binding system, antibody, or antigen-binding fragment thereof is a single polypeptide that is a chimeric antigen receptor or is contained within a chimeric antigen receptor, which is a bispecific chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain, namely 4-1BB/CD137, the alpha chain of the T cell receptor, the beta chain of the 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 transmembrane domain of the zeta chain of the T cell receptor, or any combination thereof. In some embodiments, (i) the three HCDRs of the anti-CD20 binding motif and the three LCDRs of the anti-CD20 binding motif are present in the first polypeptide, and (ii) the three HCDRs of the anti-CD19 binding motif and the three LCDRs of the anti-CD19 binding motif are together contained in a different second polypeptide. In some embodiments, the first polypeptide is a first chimeric antigen receptor or is contained in a first chimeric antigen receptor. In some embodiments, the second polypeptide is a second chimeric antigen receptor or is contained in a second chimeric antigen receptor.

In various embodiments, this disclosure includes a nucleic acid encoding at least one polypeptide of this disclosure and/or a vector containing such a nucleic acid. This disclosure further includes a method for generating engineered cells, the method comprising transfecting or transducing cells with a nucleic acid encoding at least one polypeptide of this disclosure. This document further provides cells encoding or expressing the antigen-binding system, antibody, or antigen-binding fragment provided herein, optionally said cells being immune cells, optionally said cells being T cells.

This disclosure further includes a method of treating cancer in a subject in need, 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 this disclosure. This document also provides a method of inducing an immune response in a subject or immunizing the 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 this disclosure. In some embodiments, the cells are CAR-T cells. In various implementation schemes, cancer includes acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphoblastic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell vegetations, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid 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 disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplastic syndromes and myelodysplastic syndromes, non-Hodgkin's lymphoma (NHL), plasmacytosis (including asymptomatic myeloma (accumulated multiple myeloma or indolent myeloma)). The cell therapy includes plasmablastic lymphoma, plasmacytoid dendritic cell vegetations, plasmacytomas (including plasmacytic cachexia; solitary myeloma; solitary 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 cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia (“TALL”), T-cell lymphoma, transformed follicular lymphoma or Warschitz's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, pilocellular lymphoma/leukemia, or combinations thereof. In some implementations, the cell therapy is allogeneic cell therapy or autologous cell therapy.

At least one aspect of this disclosure includes a chimeric antigen receptor comprising an anti-CD20 binding motif, said anti-CD20 binding motif comprising three heavy chain complementarity-determining regions (HCDRs) and three light chain CDRs (LCDRs), said 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 said 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) HCDR1 has a sequence according to any one of SEQ ID NO: 3-5, 25-27, 47-49, 69-71, 91-93, 113-115, 135-137, 157-159, 179-181; and 201-203; (ii) HCDR2 has According to any one of SEQ ID NO: 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184; and 204-206; (iii) HCDR3 has a sequence according to any one of SEQ ID NO: 9-11, 31-33, 53-55, 75-77, 97-99, 119-121, 141-143, 163-165, 1 (iv) LCDR1 has a sequence according to any one of 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) LCDR2 has a sequence according to any one of SEQ ID NO: 17-19, 39-41, 61-63, The sequence of any one of SEQ ID NO: 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and (vi)LCDR3 has a sequence of any one of SEQ ID NO: 20-22, 42-44, 64-66, 86-88, 108-110, 130-132, 152-154, 174-176, 196-198; and 218-220. In some implementations, the HCDR comprises: (i) HCDR1 according to any one of SEQ ID NO: 3-5; HCDR2 according to any one of SEQ ID NO: 6-8; HCDR3 according to any one of SEQ ID NO: 9-11; (ii) HCDR1 according to any one of SEQ ID NO: 25-27; HCDR2 according to any one of SEQ ID NO: 28-30; HCDR3 according to any one of SEQ ID NO: 31-33; (iii) HCDR1 according to any one of SEQ ID NO: 47-49; HCDR3 according to any one of SEQ ID NO: 5 HCDR2 of any one of SEQ ID NO: 0-52; HCDR3 of any one of SEQ ID NO: 53-55; (iv) HCDR1 of any one of SEQ ID NO: 69-71; HCDR2 of any one of SEQ ID NO: 72-74; HCDR3 of any one of SEQ ID NO: 75-77; (v) HCDR1 of any one of SEQ ID NO: 91-93; HCDR2 of any one of SEQ ID NO: 94-96; HCDR3 of any one of SEQ ID NO: 97-99; (vi) HCDR3 of any one of SEQ ID NO: 53-55; HCDR1 of any one of SEQ ID NO: 113-115; HCDR2 of any one of SEQ ID NO: 116-118; HCDR3 of any one of SEQ ID NO: 119-121; (vii) HCDR1 of any one of SEQ ID NO: 135-137; HCDR2 of any one of SEQ ID NO: 138-140; HCDR3 of any one of SEQ ID NO: 141-143; (viii) HCDR1 of any one of SEQ ID NO: 157-159; HCDR3 of any one of SEQ ID NO: 160-162 HCDR2 of any one of SEQ ID NO: 163-165; HCDR3 of any one of SEQ ID NO: 179-181; HCDR2 of any one of SEQ ID NO: 182-184; HCDR3 of any one of SEQ ID NO: 185-187; or (x) HCDR1 of any one of SEQ ID NO: 201-203; HCDR2 of any one of SEQ ID NO: 204-206; HCDR3 of any one of SEQ ID NO: 207-209; and L The CDR comprises: (i) an LCDR1 according to any one of SEQ ID NO: 14-16; an LCDR2 according to any one of SEQ ID NO: 17-19; an LCDR3 according to any one of SEQ ID NO: 20-22; (ii) an LCDR1 according to any one of SEQ ID NO: 36-38; an LCDR2 according to any one of SEQ ID NO: 39-41; an LCDR3 according to any one of SEQ ID NO: 42-44; (iii) an LCDR1 according to any one of SEQ ID NO: 58-60; an LCDR3 according to any one of SEQ ID NO: 61-63. LCDR2 according to any one of SEQ ID NO: 64-66; (iv) LCDR1 according to any one of SEQ ID NO: 80-82; LCDR2 according to any one of SEQ ID NO: 83-85; LCDR3 according to any one of SEQ ID NO: 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) LCDR3 according to SEQ I 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; LCDR3 according to any one of SEQ ID NO: 171-16 ...68-170; LCDR3 according to any one of SEQ ID NO: 171-168; LCDR2 according to any one of SEQ ID NO: 127-129; LCDR3 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 124-126; LCDR2 according to any one of SEQ ID NO: 127-129; LCDR2 according to any one of SEQ ID NO: 127-129; LCDR3 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 130-132; LCDR1 according to any one of SEQ ID NO: 1 LCDR2 of any one of SEQ ID NO: 174-176; (ix) LCDR1 of any one of SEQ ID NO: 190-192; LCDR2 of any one of SEQ ID NO: 193-195; LCDR3 of any one of SEQ ID NO: 196-198; or (x) LCDR1 of any one of SEQ ID NO: 212-214; LCDR2 of any one of SEQ ID NO: 215-217; LCDR3 of any one of SEQ ID NO: 218-220.

In some embodiments, the chimeric antigen receptor includes 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 comprise HCDR1, HCDR2, and HCDR3; the three LCDRs comprise LCDR1, LCDR2, and LCDR3; and the HCDRs and LCDRs comprise: (i) HCDR1 according to any one of SEQ ID NO:3-5; HCDR2 according to any one of SEQ ID NO:6-8; HCDR3 according to any one of SEQ ID NO:9-11; and L according to any one of SEQ ID NO:14-16. CDR1; LCDR2 according to any one of SEQ ID NO: 17-19; LCDR3 according to any one of SEQ ID NO: 20-22; (ii) HCDR1 according to any one of SEQ ID NO: 25-27; HCDR2 according to any one of SEQ ID NO: 28-30; HCDR3 according to any one of SEQ ID NO: 31-33; LCDR1 according to any one of SEQ ID NO: 36-38; LCDR2 according to any one of SEQ ID NO: 39-41; LCDR3 according to any one of SEQ ID NO: 42-44; (iii) according to HCDR1 of any one of SEQ ID NO: 47-49; HCDR2 of any one of SEQ ID NO: 50-52; HCDR3 of any one of SEQ ID NO: 53-55; LCDR1 of any one of SEQ ID NO: 58-60; LCDR2 of any one of SEQ ID NO: 61-63; LCDR3 of any one of SEQ ID NO: 64-66; (iv) HCDR1 of any one of SEQ ID NO: 69-71; HCDR2 of any one of SEQ ID NO: 72-74; HCDR3 of any one of SEQ ID NO: HCDR3 of any one of SEQ ID NO: 75-77; LCDR1 of any one of SEQ ID NO: 80-82; LCDR2 of any one of SEQ ID NO: 83-85; LCDR3 of any one of SEQ ID NO: 86-88; (v) HCDR1 of any one of SEQ ID NO: 91-93; HCDR2 of any one of SEQ ID NO: 94-96; HCDR3 of any one of SEQ ID NO: 97-99; LCDR1 of any one of SEQ ID NO: 102-104; LCDR1 of any one of SEQ ID NO: 105-107 LCDR2 according to any one of SEQ ID NO: 108-110; LCDR3 according to any one of SEQ ID NO: 113-115; HCDR1 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) LCDR3 according to any one of SEQ ID NO: 130-132; HCDR1 of any one of SEQ ID NO: 138-140; HCDR2 of any one of SEQ ID NO: 141-143; LCDR1 of any one of SEQ ID NO: 146-148; LCDR2 of any one of SEQ ID NO: 149-151; LCDR3 of any one of SEQ ID NO: 152-154; (viii) HCDR1 of any one of SEQ ID NO: 157-159; HCDR2 of any one of SEQ ID NO: 160-162; HCDR3 of any one of SEQ ID NO: 138-140; LCDR3 of any one of SEQ ID NO: 141-143; LCDR1 of any one of SEQ ID NO: 146-148; LCDR2 of any one of SEQ ID NO: 149-151; LCDR3 of any one of SEQ ID NO: 152-154; (viii) HCDR1 of any one of SEQ ID NO: 157-159; HCDR2 of any one of SEQ ID NO: 160-162; LCDR3 of any one of SEQ ID NO: 149-141; LCDR3 of any one of SEQ ID NO: 141-143; LCDR1 of any one of SEQ ID NO: 146-148; LCDR2 of any one of SEQ ID NO: 149-151; LCDR3 of any one of SEQ ID NO: 152-154; (viii) HCDR1 of any one of SEQ ID NO: 157-159; HCDR2 of any one of SEQ ID NO: 160-162; LCDR3 of any one of SEQ ID NO: 157-159; LCDR2 of any one of SEQ ID NO: 160-162; LCDR3 of any one of HCDR3 of any one of SEQ ID NO: 163-165; LCDR1 of any one of SEQ ID NO: 168-170; LCDR2 of any one of SEQ ID NO: 171-173; LCDR3 of any one of SEQ ID NO: 174-176; (ix) HCDR1 of any one of SEQ ID NO: 179-181; HCDR2 of any one of SEQ ID NO: 182-184; HCDR3 of any one of SEQ ID NO: 185-187; LCDR1 of any one of SEQ ID NO: 190-192; according to SE 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 one of SEQ ID NO: 215-217; LCDR3 according to any one of SEQ ID NO: 218-220. In various embodiments, the chimeric antigen receptor includes 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 consistent with SEQ ID NO:1, SEQ ID NO:23, SEQ ID NO:45, SEQ ID NO:67, SEQ ID NO:89, SEQ ID NO:111, SEQ ID NO:133, SEQ ID NO:155, SEQ ID NO:N O:177 or SEQ ID NO:199 is at least 80% identical; and (ii) the light chain variable structural domain is at least 80% identical to SEQ ID NO:12, SEQ ID NO:34, SEQ ID NO:56, SEQ ID NO:78, SEQ ID NO:100, SEQ ID NO:122, SEQ ID NO:144, SEQ ID NO:166, SEQ ID NO:188 or SEQ ID NO:210. In some embodiments, the chimeric antigen receptor includes 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:89. (vi) The heavy chain variable domain is at least 80% identical to SEQ ID NO:100; and the light chain variable domain is at least 80% identical to SEQ ID NO:111; (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:122. (ix) The variable domain of the heavy chain is at least 80% identical to that of SEQ ID NO:155, and the variable domain of the light chain is at least 80% identical to that of SEQ ID NO:166; or (x) The variable domain of the heavy chain is at least 80% identical to that of SEQ ID NO:177, and the variable domain of the light chain is at least 80% identical to that of SEQ ID NO:188; or (x) The variable domain of the heavy chain is at least 80% identical to that of SEQ ID NO:199, and the variable domain of the light chain is at least 80% identical to that of SEQ ID NO:210.

In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs and three LCDRs are comprised of a single polypeptide. In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs are comprised of a first polypeptide and the three LCDRs are comprised of 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, FLT3, folate-binding protein, GD2, GD3, HER1-HER2 group. The chimeric antigen receptor may contain: HER2-HER3 combination, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B cell activating factor receptor), and combinations thereof; and/or (ii) binding motifs that specifically bind to B cell characteristic antigens, optionally wherein the B cell characteristic antigen is not CD19 or CD20. In some embodiments, the chimeric antigen receptor further comprises an anti-CD19 binding motif.

In some embodiments, the chimeric antigen receptor of claim 40, wherein the anti-CD19 binding motif comprises a first domain comprising three HCDRs and a second domain comprising three LCDRs, wherein: the three HCDRs of the anti-CD19 binding motif comprise HCDR1, HCDR2, and HCDR3; the three LCDRs of the anti-CD19 binding motif comprise LCDR1, LCDR2, and LCDR3; and the HCDRs and LCDRs of the anti-CD19 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: 237-239; and LCDR3 according to any one of SEQ ID NO: 240-242. In some embodiments, the anti-CD19 binding motif comprises a first heavy chain variable domain containing three HCDRs of the anti-CD19 binding motif and a light chain variable domain containing three LCDRs of the anti-CD19 binding motif, wherein the heavy chain variable domain of the anti-CD19 binding motif is at least 80% identical to SEQ ID NO:221, and the light chain variable domain of the anti-CD19 binding motif is at least 80% identical to SEQ ID NO:232. In some embodiments, the three HCDRs of the anti-CD19 binding motif and the three LCDRs of the anti-CD19 binding motif are contained in a single polypeptide.

In some embodiments, the three HCDRs of the anti-CD20 binding motif, the three LCDRs of the anti-CD20 binding motif, the three HCDRs of the anti-CD19 binding motif, and the three LCDRs of the anti-CD19 binding motif are collectively contained in a single polypeptide. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain as 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.

In various embodiments, this disclosure includes a bicistronic chimeric antigen receptor comprising a first chimeric antigen receptor of this disclosure and a second chimeric antigen receptor comprising a binding motif that specifically binds to antigens 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, FLT3, folate-binding protein, GD 2. GD3, HER1-HER2 combination, HER2-HER3 combination, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B cell activating factor receptor), and combinations thereof; and/or binding motifs that specifically bind to B cell characteristic antigens, optionally wherein the B cell characteristic antigen is not CD19 or CD20. In some embodiments, the second chimeric antigen receptor comprises an anti-CD19 binding motif.

This disclosure further provides nucleic acids encoding at least one polypeptide of this disclosure and/or vectors containing such nucleic acids. This disclosure also includes methods for generating engineered cells, the method comprising transfecting or transducing cells with nucleic acids of this disclosure. In various embodiments, this disclosure includes cells encoding or expressing the chimeric antigen receptors provided herein, optionally said cells being immune cells, optionally said cells being T cells.

This disclosure further provides a method for treating cancer in a subject of need, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising a chimeric antigen receptor of this disclosure. This document further provides a method for inducing an immune response in a subject or immunizing the subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cell therapy compositions encoding or comprising a chimeric antigen receptor of this disclosure. In various embodiments, the cells are CAR-T cells. In various implementation schemes, cancer includes acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphoblastic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell vegetations, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid 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 disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplastic syndromes and myelodysplastic syndromes, non-Hodgkin's lymphoma (NHL), plasmacytosis (including asymptomatic myeloma (accumulated multiple myeloma or indolent myeloma)). The cell therapy includes plasmablastic lymphoma, plasmacytoid dendritic cell vegetations, plasmacytomas (including plasmacytic cachexia; solitary myeloma; solitary 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 cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia (“TALL”), T-cell lymphoma, transformed follicular lymphoma or Warschitz's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, pilocellular lymphoma/leukemia, or combinations thereof. In some implementations, the cell therapy is allogeneic cell therapy or autologous cell therapy.

In at least one aspect, this disclosure includes a chimeric antigen receptor comprising an anti-CD20 binding motif and a CD19 binding motif, wherein the anti-CD20 binding motif comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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: SEQ ID NO: 221 and 232. In some embodiments, the anti-CD20 binding motif comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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 SEQ ID NO: 221 and 232. In some embodiments, the anti-CD20 binding motif and the CD19 binding motif are comprised of a single polypeptide. In some embodiments, the anti-CD20 binding motif and the CD19 binding motif are comprised of different polypeptides.

In at least one aspect, this disclosure comprises a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, this disclosure comprises a pharmaceutical composition comprising a chimeric antigen receptor comprising an anti-CD20 binding motif having an amino acid sequence selected from the group consisting of: SEQ ID NO: 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.

Invention Details

This disclosure relates to novel polypeptides comprising neoantigen-binding molecule and polynucleotides encoding them. Some aspects of this disclosure relate to polynucleotides encoding chimeric antigen receptors (CARs) comprising at least one heavy chain and light chain (or its CDR) disclosed herein. This disclosure also provides vectors (e.g., viral vectors) comprising such polynucleotides and compositions comprising such vectors. This disclosure further provides polynucleotides encoding such CARs or TCRs and compositions comprising such polynucleotides. This disclosure additionally provides engineered cells (e.g., T cells) transduced with such polynucleotides and/or such viral vectors and compositions comprising such engineered cells. This disclosure provides compositions comprising multiple engineered T cells (e.g., pharmaceutical compositions). This disclosure provides methods for manufacturing such engineered T cells and compositions, and uses of such engineered T cells and compositions (e.g., for treating melanoma). Furthermore, this disclosure provides methods for inducing immunity against tumors, comprising administering to a subject an effective amount of cells comprising the polynucleotides, vectors, or polypeptides of this disclosure. Other aspects of this disclosure relate to CAR-containing cells and their use in T-cell therapy for treating patients with cancer.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. While this disclosure has been described in conjunction with a detailed description, the foregoing description is intended to be illustrative and not limiting of the scope of this 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 patents and scientific literature referenced herein establish 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 incorporated herein by reference. All other published references, dictionaries, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. Further features and advantages of this disclosure will be apparent from the following detailed description, including embodiments and claims.

definition

To facilitate understanding of this invention, certain terms are first defined below. Additional definitions for the following 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 indicators unless the context clearly specifies otherwise.

Unless explicitly stated or obvious in the context, as used herein, the term “or” should be understood as inclusive and encompasses both “or” and “and”.

As used herein, the term “and/or” is considered a specific disclosure of each of two specified features or components having or not having 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). Similarly, the term “and/or” as used in phrases such as “A, B and/or C” is intended to cover 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 “e.g.” and “i.e.” as used herein are used only as examples and are not intended to be restrictive, and should not be interpreted as referring only to those items explicitly listed in the specification.

Terms such as "or more," "at least," and "more than," for example, "at least one," should be understood to include, but are not 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 12 9, 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 of the stated values. This also includes any larger numbers or fractions in between.

Conversely, the term "not exceeding" includes every value less than the stated value. For example, "not exceeding 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, 5 4, 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. Also includes any smaller numbers or fractions in between.

The terms "multiple," "at least two," "two or more," and "at least the second" 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, 3 9, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 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. Also includes any larger numbers or fractions in between.

Throughout this specification, the word "comprising" or its variations shall be understood to imply that the stated element, integer, or step, or group of elements, integers, or steps, is included, but does not exclude any other element, integer, or step, or group of elements, integers, or steps. It should be understood that wherever the term "comprising" is used to describe an aspect herein, other similar aspects described as "consisting of" and/or "substantially consisting of" are also provided.

Unless explicitly stated or obvious from the context, as used herein, the term “about” refers to a value or composition within an acceptable range of error 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 “consistently of…” can mean within or greater than one standard deviation as practiced in the art. “About” or “consistently of…” can mean a range up to 10% (i.e., ±10%). Thus, “about” can be understood as 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 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to five times. When a particular value or composition is provided in this disclosure, unless otherwise stated, it should be assumed that the meaning of “about” or “consistently of” is within an acceptable margin of error for that particular value or composition.

As stated in the text, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range stated, and, where appropriate, its fractions (e.g., one-tenth and one-hundredth of an integer).

The units, prefixes, and symbols used in this document are provided in a form accepted by its International System of Units (SI). Numerical ranges include the numbers defining the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Juo, “The Concise 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”, Cammack et al. eds., 2nd ed., (2006), Oxford University Press provide a general dictionary for those skilled in the art to use many of the terms used in this disclosure.

"Administration" means the physical introduction of a pharmaceutical agent into a subject using any of the various 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 administration routes, such as by injection or infusion. The phrase "parenteral administration" refers to a method of administration other than enteral and local administration, typically by injection and, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, spinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some embodiments, the formulation is administered via non-parenteral routes, such as oral administration. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and/or over one or more long periods.

The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding molecule. Each H chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Typically, human antibodies are tetramers of approximately 150 kDa, composed of two identical heavy (H) chain polypeptides (approximately 50 kDa each) and two identical light (L) chain polypeptides (approximately 25 kDa each), which associate with each other to form a structure commonly referred to as a "Y-shape." The heavy and light chains are linked or connected by a single disulfide bond; two additional disulfide bonds link the hinge regions of the heavy chains together, thus linking the dimers together to form a tetramer. Naturally occurring antibodies are also glycosylated, for example, at the CH2 domain.

The term "human antibody" is intended to encompass antibodies having variable and constant domain sequences generated, assembled, or derived from human immunoglobulin sequences, or sequences indistinguishable from them. In some embodiments, antibodies (or antibody components) may be considered "human" even if their amino acid sequences contain residues or elements not encoded by human germline immunoglobulin sequences (e.g., variations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). The term "humanized" is intended to encompass antibodies having variable domains with sequences derived from variable domains of a non-human species (e.g., mice) modified to more closely resemble sequences encoded by human germlines. In some embodiments, "humanized" antibodies comprise one or more frame domains having amino acid sequences substantially having human frame domains, and one or more complementarity-determining regions having amino acid sequences substantially identical to those of non-human antibodies. In some embodiments, humanized antibodies comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant domain. In some implementations, the humanized antibody may comprise _CH1 , a hinge, _CH2 , _CH3 , and optionally _CH4 , a constant structural domain of the human heavy chain.

Antibodies can include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain 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, intracellular antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugated antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity molecules, Fab fragments, F(ab') ₂ fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), and any of the above antigen-binding fragments. In some embodiments, the antibodies described herein refer to polyclonal antibody groups. Antibodies can also include, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single-chain Fvs, peptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof), camel antibodies, single-chain or tandem biantibody microantibodies, ankyrin repeats or DARTs, TCR-like antibodies, microproteins, and

Immunoglobulins can be derived from any known isotype, including but not limited to IgA, secretory IgA, IgG, IgE, and IgM. IgG subtypes are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term "antibody" includes, for example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise specified, the term "antibody" also includes antigen-binding fragments or antigen-binding portions of any of the aforementioned immunoglobulins, and includes monovalent and bivalent fragments, as well as single-chain Abs.

"Antigen-binding molecule," "antigen-binding moiety," or "antibody fragment" refers to any molecule containing an antigen-binding moiety (e.g., CDR) of an antibody derived from the molecule. An antigen-binding molecule may include an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments formed from an antigen-binding molecule, dAb, linear antibodies, scFv antibodies, and multispecific antibodies. Peptide bodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of suitable antigen-binding molecules. In some embodiments, the antigen-binding molecule binds to antigens on tumor cells. In some embodiments, the antigen-binding molecule binds to antigens on cells involved in hyperproliferative diseases or binds to viral or bacterial antigens. In some embodiments, the antigen-binding molecule binds to BCMA, CLL-1, or FLT3. In some 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 to an antigen, including one or more of its complementarity-determining regions (CDRs). In a further embodiment, the antigen-binding molecule is a single-chain variable fragment (scFv). In some implementations, the antigen-binding molecule comprises or is composed of a high-affinity polymer.

In some cases, the CDR is substantially identical in sequence to a CDR found in a reference antibody (e.g., the antibody of this disclosure) and/or a CDR provided in this disclosure. In some embodiments, the CDR is substantially identical to a reference CDR (e.g., a CDR provided in this disclosure) because it is sequence-identical or contains one, two, three, four, or five (e.g., one to five) amino acid substitutions compared to the reference CDR. In some embodiments, the CDR is substantially identical to a reference CDR because it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the CDR is substantially identical to a reference CDR because it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the CDR is substantially identical to a reference CDR because, compared to the reference CDR, one amino acid is missing, added, or substituted, while the CDR has the same amino acid sequence as the reference in other respects. In some embodiments, the CDR is substantially identical to a reference CDR because, compared to the reference CDR, 2, 3, 4, or 5 (e.g., 2-5) amino acids are missing, added, or substituted, while the CDR has the same amino acid sequence as the reference CDR in other respects. In various embodiments, the antigen-binding fragment binds to the same antigen as the reference antibody.

Antigen-binding fragments can be generated in any manner. For example, in some embodiments, antigen-binding fragments can be generated enzymatically or chemically by fragmentation of a complete antibody. In some embodiments, antigen-binding fragments can be generated recombinantly (i.e., through the expression of engineered nucleic acid sequences). In some embodiments, antigen-binding fragments can be generated wholly or partially synthesized. In some embodiments, antigen-binding fragments 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” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110 to 120 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain. These sequences vary considerably between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is assumed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human frame region (FR). In certain embodiments, the variable region is a primate (e.g., a non-human primate) variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a primate (e.g., a non-human primate) frame region (FR).

The terms “VL” and “VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

Commonly used CDR definitions include: Kabat number, Chothia number, AbM number, or contact number. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on the analysis of available complex crystal structures.

Table 1: CDR Numbers

The term "Kabat number" and similar terms are recognized in the art and refer to a numbering system of amino acid residues in the variable regions of the heavy and light chains of an antibody or in its antigen-binding molecule. In some respects, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, 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, the CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In specific embodiments, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

In some respects, the CDR of an antibody can be determined according to the Chothia numbering scheme, which refers to the position of the immunoglobulin structural loop (see, for example, Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 ring is present in heavy chain amino acids 26 to 32, 33, or 34; the Chothia CDR-H2 ring is present in heavy chain amino acids 52 to 56; and the Chothia CDR-H3 ring is present in heavy chain amino acids 95 to 102. The Chothia CDR-L1 ring is present in light chain amino acids 24 to 34; the Chothia CDR-L2 ring is present in light chain amino acids 50 to 56; and the Chothia CDR-L3 ring is present in light chain amino acids 89 to 97. When using the Kabat numbering convention, the end of the Chothia CDR-HI loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). In the specific implementation, the CDR of the antibody described herein has been determined according to the Chothia numbering scheme.

The terms "constant region" and "constant domain" are interchangeable and have the meanings commonly found in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light and/or heavy chains, that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence compared to the variable domains of immunoglobulins.

When used to refer to antibodies, the term "heavy chain" is based on the amino acid sequence of constant structural domains and can refer to any different type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce IgA, IgD, IgE, IgG, and IgM antibodies, including subclasses of IgG, such as _IgG1 , _IgG2 , _IgG3 , and _IgG4 .

When used to refer to antibodies, the term "light chain" can refer to any different type based on the amino acid sequence of the constant domain, such as kappa (κ) or lambda (λ). 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). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant ( _KD ). Affinity can be measured and/or expressed in a variety of ways known in the art, including, but not limited to, the equilibrium dissociation constant ( _KD ) and the equilibrium association constant ( _KA ). _KD is calculated by the quotient of _koff / _kon , while _KA is calculated by the quotient of _kon / _koff . _kon refers, for example, the rate constant of binding between an antibody and an antigen, while _koff refers, for example, the dissociation between an antibody and an antigen. _kon and _koff can be determined by techniques known to those skilled in the art, such as KinExA.

"Conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues with 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 some embodiments, one or more amino acid residues within the CDR or framework region of an antibody or its antigen-binding molecule may be substituted with amino acid residues having similar side chains. Generally, two sequences are considered "substantially similar" if they contain conserved amino acid substitutions at the corresponding positions. For example, some amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or have "polar" or "nonpolar" side chains. Replacing one amino acid with another of the same type can be considered a conserved substitution. Exemplary amino acid classifications are summarized in Tables 2 and 3 below:

Table 2

Table 3

Polysemous amino acids 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 any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein having the amino acid sequence SEQ ID NO: 232 (e.g., the corresponding human costimulatory protein) is an amino acid that is not naturally present (i.e., not aligned with) as a wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the coding sequence of a wild-type human costimulatory protein.

The term "epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope) or an epitope can be, for example, derived from two or more discontinuous regions of a polypeptide or multiple polypeptides (conformational, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, antibody-bound epitopes can be determined by, for example, nuclear magnetic resonance spectroscopy (NMR spectroscopy), X-ray diffraction crystallography, 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., Giegé 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: Antigen crystals can be studied using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, marketed by Molecular Simulations, Inc.; see also, for example, Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.; U.S. 2004/0014194) and BUSTER (Bricogne G( Purification was performed using Acta Crystallogr D Biol Crystallogr 49 (Pt 1):37-60; Bricogne G (1997) MethEnzymol 276A:361-423, ed Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10):1316-1323. Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

If the interaction between the antigen and the first binding molecule, antibody, or antigen-binding molecule thereof blocks, restricts, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or antigen-binding molecule thereof to interact with the antigen, then the antigen-binding molecule, antibody, or antigen-binding molecule thereof "cross-competes" with the reference antibody or antigen-binding molecule thereof. Cross-competition can be complete, such as the binding of the binding molecule to the antigen completely blocking the ability of the reference binding molecule to bind to the antigen, or it can be partial, such as the binding of the binding molecule to the antigen reducing the ability of the reference binding molecule to bind to the antigen. In some embodiments, the antigen-binding molecule that cross-competes with the reference antigen-binding molecule binds to the same or overlapping epitopes 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, such as: solid-phase direct or indirect radioimmunoassay (RIA); solid-phase direct or indirect enzyme immunoassay (EIA); sandwich competitive assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); 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, 19...). 88, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling of RIA using 1-125 label (Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-antibiotin protein EIA (Cheung et al., 1990, Virology 176: 546-552); and direct labeling of 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 binding involves physical contact between entities or parts. "Indirect" binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be evaluated in any of a variety of situations, such as when studying interacting entities or parts in isolation or in more complex systems (e.g., when covalently or otherwise associated with a carrier entity and/or in biological systems such as cells).

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 antigens (e.g., epitopes or immune complexes), as those skilled in the art understand such binding. For example, molecules that specifically bind antigens may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, immunoassays, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In specific embodiments, molecules that specifically bind antigens bind to antigens with a Ka- _A ratio at least 2 log, 2.5 log, 3 log, 4 log, or greater than that when the _molecule binds to another antigen. Binding may include preferential association of a binding motif, antibody, or antigen-binding system with the target of that binding motif, antibody, or antigen-binding system compared to association with an entity that is not a target (i.e., a non-target). In some embodiments, the binding motif, antibody, or antigen-binding system selectively binds to the target if the binding to the target is greater than 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the binding to a non-target. In some embodiments, the binding motif, antibody, or antigen-binding system selectively binds to the target if the binding affinity is less than about ^10⁻⁵ M, less than about ^10⁻⁶ M, less than about ^10⁻⁷ M, less than about ^10⁻⁸ M, or less than about ^10⁻⁹ M.

In another embodiment, the antigen-specific binding molecule binds with a dissociation constant ( _Kd ) of about 1 x ^10⁻⁷ M. In some embodiments, the antigen-binding molecule binds the antigen with “high affinity” when _Kd is about 1 ^{x 10⁻⁹} M to about 5 x ^10⁻⁹ M. In some embodiments, the antigen-binding molecule binds the antigen with “very high affinity” when _Kd is about 1 x 10⁻¹⁰ M to about 5 x ^10⁻¹⁰ M. In one embodiment, the antigen-binding molecule has a _Kd of ^10⁻⁹ M. In one embodiment, the dissociation rate is less than about 1 x ^10⁻⁵ . In other embodiments, the antigen-binding molecule binds to human BCMA with a _Kd between about 1 x ^10⁻⁷ M and about 1 x ^10⁻¹³ M. In yet another embodiment, the antigen-binding molecule binds to human BCMA with a _Kd of about 1 x ^10⁻¹⁰ M to about 5 x ^10⁻¹⁰ M. In some embodiments, the antigen-binding molecule binds to human CD19, CD20, or both at a _Kd concentration between about 1 x ^{10⁻⁷ M} and about 1 x ^10⁻¹³ M. In yet another embodiment, the antigen-binding molecule binds to human CD19, CD20, or both at a _Kd concentration between about 1 x 10⁻¹⁰ M and about 5 x ^10⁻¹⁰ M.

In specific embodiments, this document provides antibodies or antigen-binding molecules thereof that bind to target human antigens (e.g., human BCMA or human CLL-1) with a higher affinity than another target antigen (e.g., non-human BCMA or non-human CLL-1). In some embodiments, this document provides antibodies or antigen-binding molecules thereof that bind to human CD19, human CD20, or both, with an affinity higher than that of another species of one or both target antigens, such as non-human CD19, non-human CD20, or both. In some embodiments, this document provides antibodies or antigen-binding molecules thereof that bind to target human antigens (e.g., human BCMA or human CLL-1) with an affinity 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, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In some embodiments, this document provides 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 that of another species of one or two target antigens, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In specific embodiments, the antibodies or antigen-binding molecules thereof described herein that bind to the target human antigen will bind to another target antigen with less than 10%, 15%, or 20% of the antibody or antigen-binding molecule bound to the human antigen, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

A "chimeric antigen receptor" or "CAR" is a molecule engineered to contain a binding motif and to activate immune cells (e.g., T cells, such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some implementations, a CAR contains a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be called CAR T cells. An "extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when present in the cell membrane, is understood to reside in the extracellular space outside the cell membrane.

"Antigen" refers to any molecule that elicits an immune response or can be bound by an antibody or antigen-binding molecule. An immune response may involve antibody production or activation of specific immune-active cells, or both. Those skilled in the art will readily understand that any macromolecule (including virtually all proteins or peptides) can act as an antigen. Antigens can be endogenously expressed, i.e., expressed from genomic DNA, or can be recombinantly expressed. Antigens may be specific to certain tissues, such as cancer cells, or they may be widely expressed. Furthermore, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen. In a specific embodiment, the antigen is all or a fragment of CD19 or CD20. "Target" is any molecule bound by a binding motif, antigen-binding system, or binder (e.g., an antibody). In some embodiments, the target is an antigen or epitope of this disclosure.

The term "neutralization" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological effects of that ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or indirectly alters the binding ability of the ligand (e.g., changes in the structure or energy of the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein it binds to from performing its biological function.

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

The term "allogeneic" refers to any substance that originates from one individual and is 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: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenovirus vector, a baculovirus vector, an EBV vector, a lactovirus 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 under natural or artificial conditions using a variety of methods. Transformation can be achieved using any known method for inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. In some embodiments, 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 lipid transfection. In some embodiments, "transformed" cells are stably transformed because the inserted DNA is capable of replicating as a self-replicating plasmid or as part of the host chromosome. In some embodiments, transformed cells can express the introduced nucleic acid.

The term "vector" refers to a recipient nucleic acid molecule modified to contain or incorporate a provided nucleic acid sequence. One type of vector is the "plasmid," which refers to a circular double-stranded DNA molecule in which additional DNA can be attached. Another type of vector is a viral vector, in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors contain sequences that guide the expression of inserted genes, and they are operatively attached to these genes. Such vectors may be referred to herein as "expression vectors." Standard techniques can be used for the engineering of carriers, for example, as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

“Cancer” refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. “Cancer” or “cancer tissue” can include tumors. Examples of cancers that can be treated by the methods of this disclosure include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, myeloma, and other white blood cell malignancies. In some embodiments, the methods of this disclosure can be used to reduce the size of tumors, for example, those originating from the following group: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, 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, endocrine system cancers, thyroid cancer, and thyroid cancer. Parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral 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), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, 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 the aforementioned cancers. In one specific embodiment, the cancer is multiple myeloma. The specific cancer may be responsive to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancers are those that are not easily treated surgically and that either initially do not respond to chemotherapy or radiation therapy, or become unresponsive over time. These cancers further include relapsed or refractory large B-cell lymphomas following two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma following two or more lines of systemic therapy, high-grade B-cell lymphoma, and DLBCL resulting from follicular lymphoma.

As used in this article, "antitumor effect" refers to biological effects that can manifest as tumor shrinkage, reduction in tumor cell number, decrease in tumor cell proliferation, reduction in the number of metastases, increase in overall or progression-free survival, increase in life expectancy, or improvement in various tumor-related physiological symptoms. Antitumor effect can also refer to the prevention of tumor development, such as through vaccines.

As used herein, “cytokine” refers to a non-antibody protein released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be expressed endogenously by cells or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (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 pro-inflammatory 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. 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 cellular chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eosinophil chemokine, eosinophil chemokine-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus activation-regulated chemokines (TARC or CCL17).

The “therapeutic effective amount,” “effective dose,” “effective amount,” or “therapeutic effective dose” of a therapeutic agent (such as engineered CAR T cells) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease regression, evidence of which includes a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of injury or disability due to disease suffering. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as assessment in human subjects during clinical trials, assessment in animal model systems used to predict efficacy in humans, or assessment by measuring the activity of the agent in in vitro analyses.

The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a class of cytotoxic lymphocytes that represent a major component of the innate immune system. NK cells repel tumor cells and virus-infected cells. They function through apoptosis, or programmed cell death. Because they do not require activation to kill cells, they are called "natural killers." T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptor (TCR) distinguishes them from other lymphocyte types. The thymus (a specialized organ of the immune system) is primarily responsible for T cell maturation. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells (i) stem cell-like memory T _SCM cells (e.g., naïve cells) express CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibit many unique functional properties of memory cells; (ii) central memory T _CM cells express L-selectin and CCR7, they secrete IL-2, but do not secrete IFNγ or IL-4; and (iii) effector memory T cells. _EM cells, however, do not express L-selectin or CCR7, but produce effector cytokines such as IFNγ 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). They produce antibodies and antigens and perform the function of antigen-presenting cells (APCs), and upon activation through antigen-antigen interactions, transform into memory B cells. In mammals, immature B cells form in the bone marrow, from which the name B cell originates.

The term "linker" (L), "linker domain," or "linker region" refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any domain/region of the CAR of the present invention. The linker may consist of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not spatially interfere with each other. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a small RNA virus 2A-like linker, a CHYSEL sequence of porcine cyclovir (P2A), a virus (T2A), or combinations thereof, variants, and functional equivalents thereof. In other embodiments, the linker sequence may contain the Asp-Val/Ile-Glu-X-Asn-Pro-Gly. ^(2A) -Pro. ^(2B) motif (SEQ ID NO:314), which results in a cleavage between 2A glycine and 2B proline. Other adapters will be apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the invention. For example, in some embodiments, the adapter can be used to link or connect different antigen-binding systems, such as the two CARs of a bicistronic CAR. The adapter can be part of a multi-element agent that links different elements to each other. For example, a polypeptide containing two or more functional or structural domains may include a segment of amino acids that links them to each other between such domains. In some embodiments, the polypeptide containing the adapter element has an integral structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains associated with each other by the adapter. The adapter can link or connect any domain/region of the CAR disclosed herein. In some embodiments, the peptide 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 (e.g., lengths of 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 some implementations, the connector is characterized by its tendency to provide flexibility to the peptide rather than employing a rigid three-dimensional structure.

"Single-chain variable fragment", "single-chain antibody variable fragment", or "scFv" antibody refers to an antibody form that contains only variable regions of the heavy chain and light chain (linked by adaptor peptides).

The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of a cell, including but not limited to deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some embodiments, the modified cells are lymphocytes (e.g., T cells), which may be obtained from a patient or donor. Cells may be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs) incorporated into the cell's genome. Engineering typically involves artificial manipulation. For example, a polynucleotide is considered "engineered" when two or more sequences that do not exist in nature linked or joined together in that order are artificially manipulated to be directly linked or joined to each other in an engineered polynucleotide. In cases where cells are manipulated using molecular biology techniques, a cell or organism is considered "engineered" if the manipulation alters its genetic information (e.g., by introducing new genetic material not previously present, such as through transformation, somatic cell hybridization, transfection, transduction, or other mechanisms, or by altering or removing previously present genetic material, such as through substitution or deletion mutations, or through other protocols). In some embodiments, the binder is a modified lymphocyte, such as a T cell, which can be obtained from a patient or donor. Engineered cells can be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs), which are integrated into the cell's genome. Descendants of engineered polynucleotides or binders are often referred to as "engineered," even if the actual operation is performed on a prior entity. In some embodiments, "engineered" refers to an entity that has been designed and manufactured. The term "designed" refers to a pharmaceutical agent that: (i) has a structure that is artificially selected or artificially selected; (ii) is produced by a process requiring artificial intervention; and/or (iii) is different from natural substances and other known pharmaceutical agents. "T-cell receptor" or "TCR" refers to an antigen recognition molecule present on the surface of T cells. During normal T-cell development, each of the four TCR genes—α, β, γ, and δ—can be rearranged, resulting in a highly diverse range of TCR proteins.

"Immune response" refers to the action of cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damage, destruction, and/or exclusion of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells in vertebrates, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "immunotherapy" refers to treatment of a subject who has a disease or is at risk of contracting or relapsing from the disease, which is performed by means of inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. T-cell therapy may include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT ^™ ), and allogeneic T-cell transplantation. However, those skilled in the art will recognize that the conditioning methods disclosed herein will enhance the effectiveness of any transplanted T-cell therapy. Examples of T-cell therapy are described in U.S. Patent Publications 2014/0154228 and 2002/0006409, U.S. Patent No. 5,728,388, and International Publication No. WO 2008/081035.

T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell populations in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL ^™ isolation and/or apheresis. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, which is incorporated herein by reference in its entirety.

The term "engineered autologous cell therapy" (which may be abbreviated as "eACT ^™ ", also known as adoptive cell transfer) is the process of collecting a patient's own T cells and then genetically altering them to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells may be engineered to express, for example, chimeric antigen receptors (CARs) or T-cell receptors (TCRs). CAR-positive (+) T cells are engineered to express extracellular single-chain variable fragments (scFvs) specific to a particular tumor antigen, which are linked to an intracellular signaling motif comprising at least one co-stimulatory domain and at least one activation domain. The co-stimulatory domain may be derived from naturally occurring co-stimulatory domains, such as those having the amino acid sequence of SEQ ID NO:1 or variants thereof (e.g., variants having a truncated hinge domain ("THD")), while the activation domain may be derived, for example, from CD3-zeta. In some embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains. CAR scFvs can be engineered to target, for example, CD19, a transmembrane protein expressed by cells of B-cell lineages (including all normal B cells and B-cell malignancies, including but not limited to NHL, CLL, and non-T-cell ALL). In some embodiments, the CAR is engineered to express the co-stimulatory domain as a separate polypeptide chain. Examples of CAR T-cell therapies and constructs are described in U.S. Patent Publications 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, all of which are incorporated herein by reference in their entirety. “Adoptive cell therapy” or “ACT” involves the transfer of immune cells with anti-tumor activity to a subject, such as a cancer patient. In some embodiments, ACT is a therapeutic approach involving the use of lymphocytes with anti-tumor activity (e.g., engineered lymphocytes).

"Patient" includes anyone with cancer (such as lymphoma or leukemia). In this article, the terms "subject" and "patient" are used interchangeably.

The term "in vitro" refers to events occurring in an artificial environment, such as in test tubes, reaction vessels, cell cultures, etc., rather than within a multicellular organism. The term "in vitro cell" refers to any cell cultured in vitro. Specifically, in vitro cells can include T cells. The term "in vivo" refers to events occurring within a multicellular organism, such as a human or non-human animal.

An "antigen-specific target region" (ASTR) refers to a CAR region that targets a specific antigen. The CAR of this invention comprises at least two target regions targeting at least two different antigens. In one embodiment, the CAR comprises three or more target regions targeting at least three or more different antigens. The target regions on the CAR are extracellular. In some embodiments, the antigen-specific target regions comprise antibodies or their functional equivalents or fragments or derivatives thereof, and each target region targets a different antigen. Target regions may comprise full-length heavy chains, Fab fragments, single-chain Fv (scFv) fragments, bivalent single-chain antibodies, or biantibodies, each of which is specific to the target antigen. However, many alternatives to promote immune responses are available, such as linked cytokines (which lead to recognition of cells with cytokine receptors), affinities, 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 this invention. Indeed, as those skilled in the art will understand, virtually any molecule that binds to a given antigen with high affinity can be used as an antigen-specific target region.

"Antigen-presenting cells" or "APCs" refer to cells that process antigens and present them to T cells. 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 compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids and there is no limit to the maximum number of amino acids that can comprise the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together 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 have many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, “stimulus” refers to a primary response induced by the binding of a stimulating molecule to its associated ligand, where the binding mediates signal transduction events. “Stimulating molecule” refers to a molecule on T cells, such as the T cell receptor (TCR)/CD3 complex that specifically binds to an associated stimulating ligand present on antigen-presenting cells. “Stimulating ligand” refers to a ligand present on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, etc.) that can specifically bind to stimulating molecules on T cells, thereby mediating primary T cell responses (including but not limited to activation, initiation of an immune response, proliferation, etc.). Stimulating ligands include, but are not limited to, anti-CD3 antibodies (e.g., OKT3), peptide-loaded MHC class I molecules, hyperactive anti-CD2 antibodies, and hyperactive anti-CD28 antibodies.

As used in this article, a “co-stimulatory signal” refers to a signal that, when combined with a primary signal (such as TCR/CD3 linkage), leads to a T cell response (such as, but not limited to, the upregulation or downregulation of proliferation and/or key molecules).

As used herein, “co-stimulatory ligands” include molecules on antigen-presenting cells that specifically bind to associated co-stimulatory molecules on T cells. Binding of co-stimulatory ligands provides signals that mediate T cell responses, including but not limited to proliferation, activation, and differentiation. Co-stimulatory ligands induce signals in addition to primary signals provided by stimulatory molecules, such as those provided by the binding of the T cell receptor (TCR)/CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Co-stimulatory ligands may include, but are not limited to, 3/TR6, 4-1BB ligands, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligands, CD40, CD7, CD70, CD83, herpesvirus invasion mediators (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible co-stimulatory ligands (ICOS-L), intracellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin beta receptors, MHC class I chain-associated protein A (MICA), MHC class I chain-associated protein B (MICB), OX40 ligands, 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).

"Co-stimulatory molecules" are associated binding partners that specifically bind to co-stimulatory ligands on T cells, thereby mediating co-stimulatory responses of T cells (e.g., but not limited to proliferation). Co-stimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, etc. CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96(Tactile), CDl-la, CDl -lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1(CD226), Fc gamma receptor, GADS, GITR, HVEM(LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITG1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a/CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof.

In this article, the terms “reduction” and “reduction” are used interchangeably and refer to any change less than the original. “Reduction” and “reduction” are relative terms and need to be compared between before and after the measurement. “Reduction” and “reduction” include complete depletion.

The terms “improvement,” “increase,” “suppression,” and “reduction” refer to values relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be included in otherwise equivalent conditions in the absence of a drug or treatment (e.g., before and/or after), or in certain systems (e.g., in the presence of an appropriate equivalent reference agent). In some embodiments, an appropriate reference measurement may be included in equivalent systems that are known or expected to respond in an equivalent manner in the presence of a relevant drug or treatment.

"Treatment" or "treatment" of a subject refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to the subject with the aim of reversing, alleviating, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions, or disease-related biochemical indicators. In one embodiment, "treatment" or "treatment" includes partial remission. In another embodiment, "treatment" or "treatment" includes complete remission. In some embodiments, treatment may be directed to subjects who do not exhibit signs of the relevant disease, condition, and/or illness, and/or subjects who exhibit only early signs of the disease, condition, and/or illness. In some embodiments, such treatment may be directed to subjects exhibiting one or more definitive signs of the relevant disease, condition, and/or illness. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, condition, and/or illness. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors statistically associated with the relevant disease, condition, and/or illness.

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

Two events or entities are “associated” with each other if the existence, level, and/or form of one event or entity is related to another. For example, if the existence, level, and/or form of an entity (e.g., a polypeptide, genetic signature, metabolite, microorganism, etc.) is associated with the occurrence and/or susceptibility to a disease, symptom, or condition (e.g., in a relevant population), then the entity is considered associated with the disease, symptom, or condition. For example, if two or more entities interact directly or indirectly such that they are physically close to each other and/or remain physically close (e.g., bind), then they are physically “associated” with each other. In other instances, two or more entities that are physically associated with each other are covalently connected or linked, or non-covalently associated, such as through hydrogen bonds, van der Waals interactions, hydrophobic interactions, attractive forces, and combinations thereof.

The term "identity" refers to the overall relevance between aggregate molecules, such as nucleic acid molecules (e.g., DNA and/or RNA molecules) and/or polypeptide molecules. Methods for calculating the percentage identity between two provided polypeptide sequences are known. The calculation of the percentage identity between two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second sequences for optimal alignment, and dissimilar sequences can be ignored for comparison purposes). 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., a nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of vacancies and the length of each vacancy (which may need to be introduced to achieve optimal alignment of the two sequences). The comparison or alignment of sequences and the determination of the percentage identity between two sequences can be accomplished using mathematical algorithms such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "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 the percentage of identity, sequences are typically aligned in a way that maximizes the match between them. An example of a computer program that can be used to determine the percentage of identity is the GCG package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wisconsin). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percentage of identity is to be determined. The sequences are aligned to achieve the best possible match of their respective amino acids or nucleotides (e.g., the "matched span" determined by the algorithm). In some implementations, the algorithm also uses standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352, for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919, for the BLOSUM62 comparison matrix). Other algorithms can 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, nick BLAST, and PSI-BLAST for amino acid sequences. Exemplary programs of this kind are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Aci See ds 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. Besides identifying similar sequences, the above procedures typically 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 in the corresponding segments of the residues are similar and/or identical. In some embodiments, the corresponding segments are complete sequences. In some implementations, the corresponding segment comprises 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 with basic sequence similarity can be homologs of each other.

"Combination therapy" refers to a situation where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more treatment components). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "dose" of the first regimen are administered before any dose of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may involve administering one or more agents or methods to a subject who is receiving other agents or methods in the combination. For clarity, combination therapy does not require the individual agents to be administered together in a single composition (or even simultaneously), although in some embodiments, two or more agents or their active portions may be used together in the combination composition, or even in the combination compound (e.g., as part of a single chemical complex or covalent entity).

The term "corresponds 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, monomeric residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as "corresponding to" residues in an appropriate reference polymer. For example, for simplicity, a canonical numbering system based on a reference-related polypeptide can be used to specify residues in a polypeptide such that the amino acid "corresponding to" the residue at position 100 does not actually have to be the 100th amino acid in the amino acid chain, as 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 BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which can be used, for example, to identify “corresponding” residues in peptides and/or nucleic acids according to this disclosure.

The term "domain" refers to a portion of an entity. In some embodiments, a "domain" is associated with structural and/or functional characteristics of an entity, such that it substantially or completely retains these structural and/or functional characteristics when physically separated from the remainder of its parent entity. 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 imparts one or more structural and/or functional characteristics to the recipient entity, such as those it possesses in the parent entity. In some embodiments, a domain is a portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a portion of a polypeptide; in some such embodiments, the domain is characterized by structural elements (e.g., amino acid sequences or sequence motifs, α-helix features, β-sheet features, coil-coil features, random coil features, etc.) and/or functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signal transduction activity, etc.).

The term "dosage form" can be used to refer to a physically discrete unit of an active agent (e.g., an antigen-binding system or antibody) intended for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is an amount (or all fractions thereof) suitable for administration of a unit dose according to a dosing regimen that has been determined to be associated with a desired or beneficial outcome when administered to a relevant population. The total amount of therapeutic composition or agent administered to a subject is determined by one or more practicing physicians 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 multiple doses, wherein each dose is time-separated from the other doses. In some embodiments, the dosing regimen comprises multiple doses and consecutive doses are spaced apart from each other by time intervals 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 intervals of different lengths. In some embodiments, all doses within the dosing regimen have the same unit dose amount. In some embodiments, different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose having a first dose amount, followed by one or more additional doses having a second dose amount different from the first dose amount. In some embodiments, the dosing regimen is periodically adjusted to achieve a desired or beneficial result.

"Effective function" refers to the biological outcome of the interaction between the antibody's Fc region and its 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). Effector functions can be antigen-dependent, antigen-independent, or both. ADCC refers to the lysis of antibody-bound target cells by immune effector cells. Without being bound by any particular 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 antigens on their surface). Effector cells mediating ADCC can include immune cells, including, but not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

"Effective cells" are immune system cells that express one or more Fc receptors and mediate one or more effector functions. In some embodiments, 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. Effector cells can be from 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc.

A "fragment" or "part" of a material or entity as described herein has a structure that includes discrete parts of a whole, such as discrete parts of a physical entity or abstract entity. In some embodiments, a fragment lacks one or more parts found in the whole. In some embodiments, a fragment consists of or contains characteristic structural elements, domains, or parts found in the whole. In some embodiments, the polymer fragment comprises, or is composed 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 monomer units (e.g., residues) found in the whole polymer. In some embodiments, the polymer fragment comprises, or is composed 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%) found in the whole polymer. In some embodiments, the whole 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 containing at least two segments. Typically, a polypeptide containing at least two such segments is considered a fusion polypeptide if the two segments are (1) portions that are not naturally present in the same peptide, and/or (2) portions that were not previously linked or connected in a single polypeptide, and/or (3) portions that were linked or connected to each other through artificial means.

The terms “gene product” or “expression product” generally refer to RNA transcribed from a gene (before and/or after processing) or polypeptides encoded by RNA transcribed from a gene (before and/or after modification).

The term “separated” means a substance that (1) has been separated from at least some of the components with which it was associated at an earlier time or with which it will otherwise be associated, and/or (2) is present in a composition containing one or more known or unknown contaminants in limited or limited amounts or concentrations. In some embodiments, the separated substance 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-material components (which associated with the substance at an earlier time, such as other components or contaminants that the substance previously associated with or will otherwise associate with). In some cases, the substance is separated if it is present in a composition containing limited or reduced amounts or concentrations of the same or similar types of molecules. For example, in some cases, if nucleic acid, DNA, or RNA material is present in a composition containing a limited or reduced amount or concentration of non-substance nucleic acid, DNA, or RNA molecules, then the nucleic acid, DNA, or RNA material is isolated. Similarly, in some cases, if polypeptide material is present in a composition containing a limited or reduced amount or concentration of non-substance polypeptide molecules, then the polypeptide material is isolated. In some embodiments, the amount can be, for example, an amount measured relative to the amount of the desired substance present in the composition. In some embodiments, the limited amount can be an amount not exceeding 100% of the amount of the substance in the composition, for example, not exceeding 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the amount of the substance in the composition (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some cases, the composition is pure or substantially pure with respect to the selected substance. In some embodiments, the separated substance 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%). If a substance is substantially free of other components or contaminants, it is considered "pure". In some embodiments, a substance may still be considered "separated" or even "pure" after being combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of separation or purity of the substance without such carriers or excipients is calculated.

“Nucleic acid” refers to any polymeric chain of nucleotides. Nucleic acid can 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 the following methods: isolation from a natural source, enzymatic synthesis (in vivo or in vitro) based on complementary template polymerization, 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 The nucleic acid may have lengths of 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 entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element encoding a polypeptide or as a complement to the sequence encoding a polypeptide.

"Operationally connected" means juxtaposed, where the described components are in a relationship that allows them to function in the intended manner. For example, "operationally connected" to a control element of a functional element is associated in a manner that enables the expression and/or activity of the functional element under conditions compatible with the control element.

The term "pharmaceutically acceptable" means a molecule or composition that, when administered to a recipient, is harmless to the recipient or provides a benefit to the recipient that outweighs any adverse effects. Regarding carriers, diluents, or excipients used to formulate compositions as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and be harmless to the recipient, or provide a benefit to the recipient that outweighs any adverse effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in carrying or transporting a drug 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 compatibility with the other components of the formulation and harmlessness to the patient, or the benefit to the recipient must outweigh any adverse 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 gum powder; malt; gelatin; talc; 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; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and/or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together 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 relevant subjects or populations, demonstrates 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 a drench (aqueous or non-aqueous solution or suspension), tablets, such as those for oral, sublingual, and systemic absorption, pills, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, such as sterile solutions or suspensions, or sustained-release formulations; topical application, such as as a cream, ointment, controlled-release patch, or spray to the skin, lungs, or mouth; intravaginal or rectal application, such as as a vaginal suppository, cream, or foam; sublingual; ocular; transdermal; or nasal, lung, and other mucosal surfaces.

The term "reference" describes a standard or control with which comparison is made. For example, in some embodiments, the agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control that serves as the agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and/or determined substantially simultaneously with the test, measurement, or determination 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 settings comparable to the object of evaluation. Sufficient similarity exists to justify reliance on and/or comparison with the selected reference or control.

Regulatory T cells ("Tregs", "Treg cells", or "Tregs") are CD4+ T lymphocyte lineages involved in controlling certain immune activities, such as autoimmunity, allergic reactions, and responses to infection. Regulatory T cells can modulate the activity of the T cell population and can also influence certain cell types of the innate immune system. Tregs can be identified by the expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Tregs typically comprise 5-10% of peripheral CD4+ T lymphocytes. However, in the tumor microenvironment (i.e., tumor-infiltrating Tregs), Tregs can account for 20-30% of the total CD4+ T lymphocyte population.

The term "sample" generally refers to an aliquot of 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 cells or organisms, such as cell populations, tissues, or animals (e.g., humans). In some embodiments, the source of interest comprises biological tissues or fluids. In some embodiments, biological tissues or fluids may comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum, semen, serum, smegma, phlegm, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomitus, and/or combinations or components thereof. In some embodiments, biological fluids may comprise intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and/or transcellular fluid. In some embodiments, the biofluid may comprise plant exudate. In some embodiments, biological tissues or samples may be obtained, for example, by aspiration, biopsy (e.g., fine-needle or tissue biopsy), swabs (e.g., oral, nasal, skin, or vaginal swabs), scraping, surgery, washing, or irrigation (e.g., bronchoalveolar, catheter, nasal, ocular, oral, uterine, vaginal, or other rinsing or irrigation). In some embodiments, the biological sample comprises cells obtained from an individual. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, 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 agents thereto) a primary sample. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques such as nucleic acid amplification or reverse transcription, separation and/or purification of certain components, etc.

The term "cancer staging" refers to a qualitative or quantitative assessment of the level of cancer progression. In some implementations, the criteria used to determine cancer staging may include, but are not limited to, one or more of the following: the location of the cancer in the body, tumor size, whether the cancer has spread to lymph nodes, and whether the cancer has spread to one or more different sites in the body. In some implementations, the so-called TNM system may be used to stage cancer, according to which T refers to the size and extent of the primary tumor (often called 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 implementations, cancer may be referred to as stage 0 (abnormal cells are present but have not spread to nearby tissues, also known as carcinoma in situ or CIS; CIS is not cancer but may become cancerous), stages I-III (cancer is present; the higher the number, the larger the tumor and the greater the extent of spread to nearby tissues), or stage IV (cancer has spread to distant sites in the body). In some implementations, cancer can be assigned to a stage selected from the following group: in situ; localized (the cancer is confined to the place where it began, with no signs of spread); regional (the cancer has spread to nearby lymph nodes, tissues, or organs); distant (the cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the stage).

The phrase "therapeutic agent" can refer to any agent that, when administered to an organism, causes the desired pharmacological effect. 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 can be a model organism or a group of human subjects. In some embodiments, a suitable population can be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical condition, or based on the presence or absence of biomarkers. In some embodiments, a therapeutic agent is a substance that can be used to reduce, improve, alleviate, inhibit, prevent, or delay the onset of one or more symptoms or features of a disease, symptom, and/or condition, reduce its severity, and/or decrease its incidence. In some embodiments, a therapeutic agent is an agent that has been or requires approval by a government agency before it can be marketed for human use. In some embodiments, a therapeutic agent is an agent that requires a medical prescription for human administration.

Various aspects of this disclosure are further described in detail in the following subsections. This disclosure provides antigen-binding systems and binders comprising at least an anti-CD20 binding motif. This disclosure provides methods and compositions for treating cancer and/or for initiating or modulating an immune response. In some embodiments, this disclosure comprises dual-targeting antigen-binding systems and binders because they comprise an anti-CD20 binding motif and a second binding motif targeting a second antigen or epitope. In some cases, the second binding motif selectively binds CD19. In various embodiments, one or more binding motifs are scFv. Exemplary binding motif amino acid sequences and nucleic acid sequences encoding them are provided herein. In some embodiments, the antigen-binding system of this disclosure is a chimeric antigen receptor. In some embodiments, the antigen-binding system of this disclosure is a bispecific or bicistronic chimeric antigen receptor. In some embodiments, the binder of this disclosure is an engineered T-cell receptor.

Various embodiments of this disclosure provide vectors encoding binding motifs or antigen-binding systems provided herein, such as vectors encoding anti-CD20/anti-CD19 antigen-binding systems, such as bispecific or bicistronic anti-CD20/anti-CD19 chimeric antigen receptors. Various embodiments of this disclosure provide conjugates that are cells encoding or expressing antigen-binding systems or binding motifs provided herein, such as T cells engineered to encode or express anti-CD20/anti-CD19 chimeric antigen receptors, such as bispecific or bicistronic anti-CD20/anti-CD19 chimeric antigen receptors. This disclosure provides conjugates, for example, those comprising immune cells genetically modified with an integrative gene, such as a nucleotide sequence of interest (e.g., a constitutive expression construct and/or an inducible expression construct containing such a nucleotide sequence). In some embodiments, this disclosure provides a method of treating a subject with a tumor, comprising administering to the subject the conjugate therapy and/or the protein therapeutic agent described herein. In some embodiments, the method further includes administering one or more additional therapies (e.g., the second binder described herein (e.g., CAR-T cells, CAR-NK cells, TCR-T cells, TIL cells, allogeneic NK cells, and autologous NK cells)), antibody-drug conjugates, antibodies, bispecific antibodies, T cell-conjugating bispecific antibodies, engineered antibodies, and/or peptides).

Other features, objects, and advantages of this disclosure will become apparent in the following detailed description. However, it should be understood that while the detailed description indicates embodiments of this disclosure, it is given by way of illustration only and not limitation.

The anti-CD20 binding motif of this disclosure may comprise an antigen-binding sequence as found in the antibodies described herein. In some instances, the anti-CD20 binding motif of this disclosure comprises an antigen-binding fragment as described herein. Unless otherwise stated, references to CD20 in this disclosure should be understood to refer to human CD20. In various embodiments, the anti-CD20 binding motif of this disclosure comprises at least one heavy chain CDR (HCDR) provided herein, such as at least one HCDR disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two HCDRs provided herein, such as at least two HCDRs disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three HCDRs provided herein, such as three HCDRs disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises at least one light chain CDR (LCDR) provided herein, such as at least one LCDR disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two LCDRs provided herein, such as at least two LCDRs disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three LCDRs provided herein, such as the three LCDRs disclosed in any of Tables 4-13.

In various embodiments, the anti-CD20 binding motif of this disclosure comprises at least one HCDR provided herein, such as at least one HCDR disclosed in any of Tables 4-13, and at least one LCDR provided herein, such as at least one LCDR disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises one HCDR provided herein, such as at least one HCDR disclosed in any of Tables 4-13, and one LCDR provided herein, such as derived from the tables in Tables 4-13 identical to the HCDRs. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two HCDRs provided herein, such as at least two HCDRs disclosed in any of Tables 4-13, and two LCDRs provided herein, such as at least two LCDRs disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two HCDRs provided herein, such as at least two HCDRs disclosed in any of Tables 4-13, and two LCDRs provided herein, such as derived from the tables in Tables 4-13 identical to the HCDRs. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three HCDRs provided herein, such as the three HCDRs disclosed in any of Tables 4-13, and three LCDRs provided herein, such as the three LCDRs disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three HCDRs provided herein, such as the three HCDRs disclosed in any of Tables 4-13, and three LCDRs derived from the tables in Tables 4-13 that contain the same HCDRs.

In various embodiments, the anti-CD20 binding motif of this disclosure comprises at least one heavy chain frame region (heavy chain FR) of the heavy chain variable domain disclosed herein, such as at least one heavy chain FR chain of the heavy chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two heavy chain FRs of the heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of the heavy chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three heavy chain FRs of the heavy chain variable domain disclosed herein, such as three heavy chain FRs of the heavy chain variable domain disclosed in any of Tables 4-13.

In various embodiments, the anti-CD20 binding motif of this disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, such as at least one light chain FR of a light chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, such as at least two light chain FRs of a light chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, such as three light chain FRs of a light chain variable domain disclosed in any of Tables 4-13.

In various embodiments, the anti-CD20 binding motif of this disclosure comprises at least one heavy chain FR of a heavy chain variable domain disclosed herein, such as at least one heavy chain FR of a heavy chain variable domain disclosed in any of Tables 4-13, and at least one light chain FR of a light chain variable domain disclosed herein, such as at least one light chain FR of a light chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises one heavy chain FR of a heavy chain variable domain disclosed herein, such as at least one heavy chain FR of a heavy chain variable domain disclosed in any of Tables 4-13, and one light chain FR of a light chain variable domain disclosed herein, such as derived from the tables in Tables 4-13 that are identical to the heavy chain FR. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two heavy chain FRs of the heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of the heavy chain variable domain disclosed in any of Tables 4-13, and two light chain FRs of the light chain variable domain disclosed herein, such as at least two light chain FRs of the light chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two heavy chain FRs of the heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of the heavy chain variable domain disclosed in any of Tables 4-13, and two light chain FRs of the light chain variable domain disclosed herein, such as those derived from the tables in Tables 4-13 that are identical to the heavy chain FRs. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three heavy chain FRs of the heavy chain variable domain disclosed herein, such as the three heavy chain FRs of the heavy chain variable domain disclosed in any of Tables 4-13, and three light chain FRs of the light chain variable domain disclosed herein, such as the three light chain FRs of the light chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises three heavy chain FRs of the heavy chain variable domain disclosed herein, such as the three light chain FRs of the light chain variable domain disclosed in any of Tables 4-13, and three light chain FRs derived from the tables in Tables 4-13 that are identical to the heavy chain FRs.

The exemplary antibody sequences provided in Table 4-13 are applicable to any antibody form, including, for example, tetrameric antibodies, monospecific antibodies, bispecific antibodies, antigen-binding fragments, or binding motifs. The heavy chain variable domains and light chain variable domains, and portions thereof, provided in Table 4-13 can be included in the binding motif.

In various embodiments, the anti-CD20 binding motif of this disclosure comprises one, two, or three FRs that, together or individually, share at least 75% identity with the FR of the corresponding heavy chain variable domain of any of the heavy chain variable domains disclosed in any of Tables 4-13 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In various embodiments, the anti-CD20 binding motif of this disclosure comprises one, two, or three FRs that, together or individually, share at least 75% identity with the FR of the corresponding light chain variable domain of any of the light chain variable domains disclosed in any of Tables 4-13 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%).

In various embodiments, the anti-CD20 binding motif of this 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%) with respect to any of the heavy chain variable domains disclosed in Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two heavy chain variable domains, each having at least 75% sequence identity with a heavy chain variable domain disclosed in any of Tables 4-13 (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%), said heavy chain variable domains may be the same or different.

In various embodiments, the anti-CD20 binding motif of this 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%) with respect to the light chain variable domain disclosed in any of Tables 4-13. In various embodiments, the anti-CD20 binding motif of this disclosure comprises two light chain variable domains, each having at least 75% sequence identity with a light chain variable domain disclosed in any of Tables 4-13 (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%), said light chain variable domains may be the same or different.

In various embodiments, the anti-CD20 binding motif of this 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%) with any of the heavy chain variable domains disclosed in 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%) with any of the light chain variable domains disclosed in Tables 4-13. In various embodiments, the anti-CD20 binding motif of this 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%) with any of the heavy-chain variable domains disclosed in Tables 4-13, and ...100%, or 95-100%) with any of the heavy-chain variable domains disclosed in Tables 4-13, and The light chain variable structural domains disclosed in any of 4-13 have 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%), and the heavy chain variable structural domains and light chain variable structural domains are optionally derived from the same tables in Tables 4-13.

In various embodiments, the anti-CD20 binding motif of this 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%) with respect to the heavy chain variable domains disclosed in any 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%) with respect to the light chain variable domains disclosed in any of Tables 4-13. The light chain variable structural domains are 0%, at least 95%, or 100% identical; for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% identical, wherein in various embodiments, (i) each heavy chain variable structural domain may be the same or different; (ii) each light chain variable structural domain may be the same or different; (iii) at least one heavy chain variable structural domain and at least one light chain variable structural domain may be derived from the same table in Tables 4-13; or (iv) both heavy chain variable structural domains and both light chain variable structural domains are derived from the same table in Tables 4-13. Each of the heavy chain variable domain and light chain variable domain sequences in Tables 4-13 represents an exemplary antibody and comprises (i) the heavy chain variable domain of the exemplary antibody; (ii) the DNA sequence encoding the 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) the light chain variable domain of the exemplary antibody; (v) the DNA sequence encoding the 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 sequence, as well as the nucleotide sequence encoding each CDR amino acid sequence and the nucleotide sequence encoding the corresponding FR amino acid sequence.

In various embodiments, the binding motif may include the heavy chain variable domain of this disclosure (e.g., having at least 75% sequence identity with the heavy chain variable domains in any of Tables 4-13, such as at least 80%, 85%, 90%, 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), and the light chain variable domain of this disclosure (e.g., having at least 75% sequence identity with the light chain variable domains in any of Tables 4-13). The identity of the column, for example, at least 80%, 85%, 90%, 95% or 100% identity; for example 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) and the connector (for example, the connector according to SEQ ID NO:247 and/or the connector according to any one of SEQ ID NO:307-313; see, for example, Whitlow et al. Protein Eng. 1993 Nov; 6(8):989-95). In various embodiments, the binding motif may include a leader sequence, a heavy chain variable domain of the present disclosure (e.g., having at least 75% sequence identity with any of the heavy chain variable domains in Tables 4-13, such as at least 80%, 85%, 90%, 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), a light chain variable domain of the present disclosure (e.g., having at least 75% sequence identity with any of the light chain variable domains in Tables 4-13, such as at least 80%, 85%, 90%, 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), and a linker. If an amino acid or nucleotide sequence comprising a binding motif comprising the heavy chain variable domain and the light chain variable domain of this disclosure is provided, the linker conjugating these two variable domains will be readily apparent from the sequence based on this disclosure. If an amino acid or nucleotide sequence comprising a binding motif comprising the heavy chain variable domain and the light chain variable domain of this disclosure is provided, the leader sequence will be readily apparent from this disclosure. For the avoidance of doubt, the heavy chain variable domain and the light chain variable domain of the present invention may be present in any orientation, for example, wherein the heavy chain variable domain is at the C-terminus of the light chain variable domain or wherein the heavy chain variable domain is at the N-terminus of 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 additional linkers.

In some embodiments, the anti-CD20 binding motif of this disclosure comprises a binding motif that includes the heavy chain variable domain of this disclosure, the light chain variable domain of this disclosure, and a connector having at least 75% sequence identity with SEQ ID NO:247 (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%). In some embodiments, the anti-CD20 binding motif of this disclosure comprises a binding motif that includes a connector according to SEQ ID NO:247 and/or a connector according to any one of SEQ ID NO:307-313. In some embodiments, the anti-CD20 binding motif of this disclosure comprises a binding motif that includes the heavy chain variable domain of this disclosure, the light chain variable domain of this disclosure, and a leader sequence having at least 75% sequence identity with SEQ ID NO:245 (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%). In some embodiments, the anti-CD20 binding motif of this disclosure comprises a binding motif that includes the CSF2RA leader sequence according to SEQ ID NO:245. In some embodiments, the anti-CD20 binding motif of this disclosure comprises a binding motif that includes the heavy chain variable domain of this disclosure, the light chain variable domain of this disclosure, the linker of this disclosure, and the leader sequence of this disclosure. Exemplary nucleotide sequences encoding anti-CD19 binding motifs and their components are found in SEQ ID NO: 246 and 248. In various embodiments, the binding motif of this disclosure has a sequence according to any one of the sequences in Table 53 (SEQ ID NO: 251-260).

The binding agents of this disclosure based on the exemplary antibodies provided herein (such as Ab1, for example) may be provided in any fragment or form, comprising a heavy chain variable domain according to the exemplary antibody shown and a light chain variable domain according to the exemplary antibody shown.

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)

This disclosure includes an antibody and antigen binding system comprising an anti-CD20 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., CD19)). Dual-target antigen binding systems include bispecific CARs and bicistron CARs. Many antigen binding motifs are known. In various embodiments, the second target antigen is CD19. This specification contains a variety of secondary target antigens, including but not limited to 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 HER1-HER2, bound HER2-HER3, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1 or VEGFR2 or combinations thereof as secondary antigens. Therefore, in various embodiments, the antigen-binding system or antibody of this disclosure may comprise a first binding motif as an anti-CD20 binding motif and a binding motif as 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, FL The second binding motifs of a second antigen, including T3, folate-binding protein, GD2, GD3, bound HER1-HER2, bound HER2-HER3, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, or VEGFR2, or combinations thereof. In some embodiments, the second antigen is a characteristic antigen of B cells or their subgroups, optionally wherein the second antigen is not CD19 or CD20. Examples of binding motifs targeting these second antigens are known and/or provided herein.

In some cases, in antigen-binding systems such as bispecific CARs containing an anti-CD20 binding motif (e.g., containing the heavy chain variable domain and/or light chain variable domain of this disclosure) and an anti-CD19 binding motif (e.g., containing the heavy chain variable domain and/or light chain variable domain of this disclosure), the anti-CD20 binding motif (or its heavy chain variable domain and/or light chain variable domain) is closer to the C-terminus of the chimeric antigen receptor than the anti-CD19 binding motif (or its heavy chain variable domain and/or light chain variable domain). In some cases, in antigen-binding systems such as bispecific CARs containing an anti-CD20 binding motif (e.g., containing the heavy chain variable domain and/or light chain variable domain of this disclosure) and an anti-CD19 binding motif (e.g., containing the heavy chain variable domain and/or light chain variable domain of this disclosure), the anti-CD20 binding motif (or its heavy chain variable domain and/or light chain variable domain) is closer to the N-terminus of the reagent than the anti-CD19 binding motif (or its heavy chain variable domain and/or light chain variable domain).

CD19 (also known as differentiation cluster 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 stated, references to CD19 in this disclosure refer to human CD19. It is found on the surface of B cells. Because CD19 expression is a marker of B cells, it can be used as an antigen, for example, to identify B cells and cancer cells derived from B cells, such as B-cell lymphoma. Anti-CD19 antibodies can bind to CD19 expressed on, 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-ALL cells, and null-cell acute lymphoblastic leukemia (NULL-ALL) cells, to provide some non-limiting examples. Exemplary pharmaceutical products, including antigen-binding systems containing anti-CD19 binding motifs, are CD19-directed, genetically modified autologous T-cell immunotherapy indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma following 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 resulting from follicular lymphoma (see FDA-approved package insert for methods and compositions relating to immunotherapy, the entire contents of which are incorporated herein by reference). Another exemplary pharmaceutical product, including an antigen-binding system containing an anti-CD19 binding motif, is a CD19-directed, genetically modified autologous T-cell immunotherapy indicated for the treatment of: (1) patients under the age of 25 years of age with refractory or second- or subsequent 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 resulting from follicular lymphoma (see the FDA-approved package insert for methods and compositions related to immunotherapy, the entire contents of which are incorporated herein by reference).

Both contain antibody-binding domains derived from anti-human CD19 antibodies. Many anti-CD19 antibodies are believed to bind to the CD19 epitope encoded in exon 4 of the CD19 gene. Other anti-CD19 binding motifs may recognize different CD19 epitopes or the same epitope with different affinities. Antigen-binding systems may contain antigen-binding domains derived from, for example, SJ25C1. A CD19 antibody, clone SJ25C1, is derived from a hybridization of Sp2/0 mouse myeloma cells with spleen cells isolated from BALB/c mice immunized with NALM1 and NALM16 cells. The SJ25C1 antigen-binding domain is used in other investigational CD19-targeted chimeric antigen receptor (CAR) T-cell therapies.

The anti-CD19 binding motif of this disclosure may comprise an antigen-binding sequence as found in the antibodies described herein. In some embodiments, the anti-CD19 binding motif of this disclosure comprises the antigen-binding fragment provided herein.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises at least one HCDR provided herein, such as at least one HCDR disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two HCDRs provided herein, such as at least two HCDRs disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises three HCDRs provided herein, such as the three HCDRs disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises at least one LCDR provided herein, such as at least one LCDR disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two LCDRs provided herein, such as at least two LCDRs disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises three LCDRs provided herein, such as three LCDRs disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises at least one HCDR provided herein, such as the at least one HCDR disclosed in Table 14, and at least one LCDR provided herein, such as the at least one LCDR disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two HCDRs provided herein, such as the at least two HCDRs disclosed in Table 14, and two LCDRs provided herein, such as the at least two LCDRs disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises three HCDRs provided herein, such as the three HCDRs disclosed in Table 14, and three LCDRs provided herein, such as the three LCDRs disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises at least one heavy chain frame region (heavy chain FR) of the heavy chain variable domain disclosed herein, such as at least one heavy chain FR chain of the heavy chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two heavy chain FRs of the heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of the heavy chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises three heavy chain FRs of the heavy chain variable domain disclosed herein, such as three heavy chain FRs of the heavy chain variable domain disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, such as at least one light chain FR of a light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, such as at least two light chain FRs of a light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, such as three light chain FRs of a light chain variable domain disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises at least one heavy chain FR of the heavy chain variable domain disclosed herein, such as at least one heavy chain FR of the heavy chain variable domain disclosed in Table 14, and at least one light chain FR of the light chain variable domain disclosed herein, such as at least one light chain FR of the light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two heavy chain FRs of the heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of the heavy chain variable domain disclosed in Table 14, and two light chain FRs of the light chain variable domain disclosed herein, such as at least two light chain FRs of the light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises three heavy chain FRs of the heavy chain variable domain disclosed herein, such as three heavy chain FRs of the heavy chain variable domain disclosed in Table 14, and three light chain FRs of the light chain variable domain disclosed herein, such as three light chain FRs of the light chain variable domain disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this disclosure comprises one, two, or three FRs that together or individually have at least 75% identity with the FRs of the corresponding heavy chain variable domains disclosed in Table 14 (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%). In various embodiments, the anti-CD19 binding motif of this disclosure comprises one, two, or three FRs that together or individually have at least 75% identity with the FRs of the corresponding light chain variable domains disclosed in Table 14 (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%).

In various embodiments, the anti-CD19 binding motif of this 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%) with respect to the heavy chain variable domains disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two heavy chain variable domains, each having at least 75% sequence identity with the heavy chain variable domains disclosed in Table 14 (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%), said heavy chain variable domains may be the same or different.

In various embodiments, the anti-CD19 binding motif of this 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%) with respect to the light chain variable domains disclosed in Table 14. In various embodiments, the anti-CD19 binding motif of this disclosure comprises two light chain variable domains, each having at least 75% sequence identity with the light chain variable domains disclosed in Table 14 (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%), said light chain variable domains may be the same or different.

In various embodiments, the anti-CD19 binding motif of this 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%) with respect to the heavy chain variable domains 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%) with respect to the light chain variable domains disclosed in Table 14.

In various embodiments, the anti-CD19 binding motif of this 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%) with the heavy-chain variable domains 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%) with the light-chain variable domains disclosed in Table 14. The variable domains are light chain variable domains 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%), 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 some embodiments, the anti-CD19 binding motif of this disclosure comprises a binding motif that includes the heavy chain variable domain of this disclosure, the light chain variable domain of this disclosure, and a linker having at least 75% sequence identity with SEQ ID NO:247 (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%). In some embodiments, the anti-CD19 binding motif of this disclosure comprises a binding motif that includes a linker according to SEQ ID NO:247 and/or a linker according to any one of SEQ ID NO:307-313. In some embodiments, the anti-CD19 binding motif of this disclosure comprises a binding motif that includes the heavy chain variable domain of this disclosure, the light chain variable domain of this disclosure, and a leader sequence having at least 75% sequence identity with SEQ ID NO:245 (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%). In some embodiments, the anti-CD19 binding motif of this disclosure comprises a binding motif that includes the CSF2RA leader sequence according to SEQ ID NO:245. In some embodiments, the anti-CD19 binding motif of this disclosure comprises a binding motif that includes the heavy chain variable domain of this disclosure, the light chain variable domain of this disclosure, the linker of this disclosure, and the leader sequence of this disclosure. In some embodiments, the binding motif has a sequence as shown in SEQ ID NO:243. Exemplary nucleotide sequences encoding anti-CD19 binding motifs and their components are found in SEQ ID NO: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, adjacent to one or more additional linkers.

Table 14: Exemplary anti-CD19 antibody sequences (Ab11)

Antigen binding systems include, for example, bispecific and bicistronic chimeric antigen receptors (CARs). This disclosure particularly provides antigen binding systems targeting CD20 and a second target antigen, such as CD19. In some embodiments, the antigen binding systems of this disclosure comprise bispecific antigen binding systems. In some embodiments, the antigen binding systems of this disclosure comprise bicistronic antigen binding systems (e.g., systems comprising a first CAR and a second CAR, wherein the first and second CARs are expressed in the same cells). A bicistronic CAR may comprise two CARs that bind different targets and are encoded by a single vector. A bicistronic CAR may comprise a first CAR containing an anti-CD19 binding motif and a second CAR containing an anti-CD20 binding motif. The binding motifs associated with various CAR frameworks are interchangeable, and the combinations of features provided in this embodiment are exemplary and not limiting. In some embodiments, the first and second CARs of a bicistronic CAR (e.g., an anti-CD20 CAR and an anti-CD19 CAR) are encoded by separate genes and/or expressed as separate mRNA molecules. In some implementations, the first and second CARs of a bicistronic CAR (e.g., anti-CD20 CAR and anti-CD19 CAR) are encoded by a single gene and/or expressed together in a single mRNA molecule, wherein the expressed protein comprises the first CAR, a cleavable adaptor domain, and the second CAR. The first and second CARs of a bicistronic CAR are typically expressed together in immune cells, such as CAR-T cells, so that a single CAR-T cell expresses a CAR targeting each target antigen (e.g., each of CD20 and CD19).

In various embodiments, bicistronic CAR vectors utilize ribosomal skipping sequences or internal ribosomal entry sites. A single vector encoding two independent CAR molecules separated by ribosomal skipping sequences can express a bicistronic CAR. In various embodiments, a bicistronic CAR comprises a first CAR and a second CAR, wherein the sequences of the first CAR and the second CAR differ only in terms of the binding motif. In various embodiments, a bicistronic CAR comprises a first CAR and a second CAR, wherein the sequences of the first CAR and the second CAR differ only in terms of 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 one or all of its components (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 contain co-stimulatory domains provided herein, such as CD28, 41BB, OX40, or ICOS co-stimulatory domains.

The CAR of a bicistronic CAR may include a binding motif, a hinge, a transmembrane domain, and an intracellular domain containing a co-stimulatory domain and an activation domain. The binding motif may be an anti-CD19 or anti-CD20 binding motif as disclosed herein. The hinge and transmembrane domain may be a 28T (CD28) domain or a CD8K domain containing both 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-CD20 and anti-CD19 binding motifs) are contained within a single bispecific CAR. In such bispecific CARs, the CAR molecule itself can 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 proximal and distal membrane binding motifs. In some embodiments, the anti-CD20 binding motif is proximal to the membrane, while the anti-CD19 binding motif is distal to the membrane. In other embodiments, the anti-CD19 binding motif is distal to the membrane, while the anti-CD20 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 selected antigens. CARs can be engineered to recognize antigens and, upon binding to those antigens, activate immune cells to attack and destroy cells carrying those antigens. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells. CARs typically contain extracellular binding motifs that mediate antigen binding (e.g., anti-CD20 and/or anti-CD19 binding motifs), transmembrane domains that cross or are understood to cross the cell membrane when the antigen-binding system is present on the cell surface or cell membrane, and intracellular (or cytoplasmic) signaling domains.

According to at least one non-limiting viewpoint, at least three “generations” of CAR compositions have existed. In first-generation CARs, a binding motif (e.g., a single-stranded variable motif) is connected or linked to a signal transduction domain (e.g., CD3ζ) via a transmembrane domain, optionally including a hinge domain and one or more spacer regions. In second-generation CARs, a co-stimulatory domain (CM1, such as CD28, 4-1BB, or OX-40) is introduced along with the signal transduction domain (e.g., CD3ζ). In third-generation CARs, a second co-stimulatory domain (CM2) is included.

TCRs are heterodimers composed of α and β chains. TCR signaling requires the recruitment of signaling proteins that generate immune synapses. Furthermore, the localization of TCRs at the plasma membrane depends on the expression of the CD3 complex in T cells. Engineered single-chain TCRs can be generated, for example, using the transmembrane and signaling domains of CAR constructs; methods and constructs for this purpose are known (e.g., sTCRs and TCR-CAR molecules, such as fusions of the TCRβ chain with CD28™ and CD28 and CD3ζ). The anti-CD20 and/or anti-CD19 antigen-binding systems of this disclosure may comprise one or more antigen-binding motifs that bind CD20 and/or CD19. 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 CD3-episilon activation domain. In some embodiments, the anti-CD20 and/or anti-CD19 antigen binding system of this disclosure comprises at least a binding motif for binding human CD20, a co-stimulatory domain, an extracellular domain, a transmembrane domain, and a CD3-zeta or CD3-episilon activation domain.

In some embodiments, the antigen-binding system of this disclosure may comprise an antigen-binding system comprising one or more of the following: a leader peptide (P), a binding motif (B), an extracellular domain (E) of a co-stimulatory protein, a transmembrane domain (T), a co-stimulatory domain (C), a second co-stimulatory domain (C'), and an activation domain (A). In some cases, the antigen-binding system is configured as: B ETA. In some cases, the antigen-binding system is configured as: P B ETA. In some cases, the antigen-binding system is configured as: B ETA. In some cases, the antigen-binding system is configured as: P B ETA. In some cases, the antigen-binding system is configured as: B ETA C'A. In some cases, the antigen-binding system is configured as: P B ETA C'A. In some embodiments, the antigen-binding system comprises VH and VL, optionally wherein the CAR is configured as follows: P-VH-VL-E-T-C-A or P-VL-VH-E-T-C-A. In some embodiments, VH and VL are linked by a linker (L), optionally wherein the CAR is configured from the N-terminus to the C-terminus as follows: 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 contain any binding motif, such as the antibodies provided in this disclosure, or the binding motifs of this disclosure. In some embodiments, the binding motif can contain an anti-CD20 binding motif and/or an anti-CD19 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 strand along with other CAR components. See, for example, U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshharet et al., Cancer Immunol Immunotherapy (1997) 45:131-136; Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797, all of which are incorporated herein by reference regarding binding motif domains in CARs. A binding motif, or scFv, is a single-stranded antigen-binding fragment comprising a heavy-chain variable domain and a light-chain variable domain connected or linked together. See, for example, U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136, all of which are incorporated herein by reference regarding the binding motif domain. When derived from parental antibodies, the binding motif may retain some, all, or substantially all of the binding between the parental antibody and the target antigen.

A hinge can be an extracellular domain of an antigen-binding system located between a binding motif and a transmembrane domain. Hinges may also be referred to as extracellular domains or "spacer regions." Hinges can contribute to receptor expression, activity, and/or stability. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain. Hinges can also provide flexibility for accessing target antigens. Hinges may include immunoglobulin-like hinge domains.

In some embodiments, the antigen-binding system of this disclosure may include a hinge, which is, is derived from, or is derived from (e.g., includes all or a fragment thereof) an immunoglobulin-like hinge domain. In some embodiments, the hinge domain is derived from or is derived from an immunoglobulin. In some embodiments, the hinge domain is selected from hinges of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or fragments thereof.

The hinge can be derived from a natural source or from a synthetic source. In some embodiments, the antigen-binding system of this disclosure may include a hinge that is, is derived from, or is derived from (e.g., includes all or fragments thereof) CD2, CD3 delta, CD3epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA2 ...49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD49d (ITGA2), CD 6) CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1) ), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160 (BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zet a), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279 (PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30) The CAR contains, but is not limited to, CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulators (ICOS), LFA-1 (CD11a/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor or Toll ligand receptor, or fragments or combinations thereof. In some embodiments, the CAR does not contain a CD28 hinge.

In some embodiments, the antigen-binding system of this disclosure may include a hinge that is, is derived from, or is derived from (e.g., includes all or a fragment thereof) a CD8 alpha hinge. In some embodiments, the hinge is, is derived from, or is derived from a CD28 hinge. In some embodiments, the hinge is a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge, wherein the fragment is anything smaller than the whole. In some embodiments, the 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 excluding 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 C-terminus. 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, the polynucleotide encoding the hinge domain comprises 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%) the same polynucleotide sequence as a known nucleotide sequence. In some embodiments, the polypeptide sequence of the hinge domain comprises 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%) the same polypeptide sequence as a known polypeptide sequence.

Generally, a "transmembrane domain" (e.g., in an antigen-binding system) refers to a domain that, when present on the cell surface or at the cell membrane (e.g., spanning part or all of the cell membrane), has the property of being present in the membrane. The co-stimulatory domain of the antigen-binding system of this 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 is present in the membrane. For example, in some embodiments, the transmembrane domain is characterized by a specific segment or portion of the protein being 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 Prosite (prosite.expasy.org) are examples of such programs.

The type of transmembrane domain included in the antigen-binding system described herein is not limited to any particular type. In some embodiments, transmembrane domains that naturally associate with binding motifs and/or intracellular domains are selected. In some cases, the transmembrane domain contains modifications (e.g., deletions, insertions, and/or substitutions) of one or more amino acids, for example, to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

Transmembrane domains can be derived from natural or synthetic sources. When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. Exemplary transmembrane domains can be derived from (e.g., may include at least one of its transmembrane domains) the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activated NK cell receptors, immunoglobulin proteins. B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, Cytokine Receptor, DAP-10, DNAM1 (CD226), Fc Gamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM- 1. Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell co-stimulatory molecule (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITG1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a/CD18), MHC class 1 molecule, N KG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or fragments, truncated or combinations thereof. In some embodiments, the transmembrane domain may be synthetic (and may, for example, primarily contain hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is contained at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly connected to or linked to the cytoplasmic domain. In some embodiments, short oligopeptide or polypeptide linkers (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 duplex.

The polynucleotide and polypeptide sequences for transmembrane domains provided herein are known. In some embodiments, the polynucleotide encoding the transmembrane domain comprises 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%) the same polynucleotide sequence as a known nucleotide sequence. In some embodiments, the polypeptide sequence of the transmembrane domain comprises 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 96%, at least 97%, at least 98%, at least 99%, or at least 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the same polypeptide sequence as a known polypeptide sequence. Optionally, short-spacer regions may form connections between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

Intracellular domains (or cytoplasmic domains) contain one or more signal transduction domains that induce and/or mediate intracellular signaling upon binding of a target antigen to a binding motif, such as activating one or more immune cell effector functions (e.g., innate immune cell effector functions). In some embodiments, the signal transduction domain of the intracellular domain mediates the activation of at least one normal effector function of an 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 signal transduction domain of the intracellular domain mediates T cell activation, proliferation, survival, and/or other T cell functions. The intracellular domain may contain a signal transduction domain as an activating domain. The intracellular domain may contain a signal transduction domain as a co-stimulatory signal transduction domain.

Intracellular signal transduction domains that can transduce signals when antigens bind to immune cells are known, and any of them may be included in the antigen-binding system disclosed herein. For example, it is known that the cytoplasmic sequence of the T cell receptor (TCR) initiates signal transduction after the TCR binds to an antigen (see, for example, Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

In some implementations, the signal transduction domain and/or activation domain contain an immune receptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signal transduction 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, for example, Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)).

In some implementations, suitable signal transduction domains include, but are not limited to, 4-1BB/CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, etc. CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R a alpha, inducible T cell costimulators (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, CD83-binding ligands, LIGHT, LIGHT, LTBR, Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1; CD1-1a/CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44 NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 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.

CARs can include co-stimulatory signal transduction domains, for example, to increase signal transduction efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (ibid.), Song et al., Blood 119:696-706 (2012); 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). Signaling generated by TCRs alone may not be sufficient to fully activate T cells, while secondary or co-stimulatory signals can increase activation. Therefore, in some embodiments, the signal transduction domain further includes one or more additional signal transduction domains (e.g., co-stimulatory signal transduction domains) that activate one or more immune cell effector functions (e.g., the innate immune cell effector functions described herein). In some embodiments, a portion of such a co-stimulatory signal transduction domain may be used, provided that portion transduces effector signaling. In some embodiments, the cytoplasmic domain described herein includes 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 a ligand that binds to CD83. Exemplary co-stimulatory proteins have the amino acid sequence of a co-stimulatory protein naturally present on T cells, the complete natural amino acid sequence of which is described in NCBI reference sequence: NP_006130.1. In some cases, the CAR contains a 41BB co-stimulatory domain encoded by the sequence according to SEQ ID NO:270, as shown below:

SEQ ID NO:270

AGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGG ACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat

The polynucleotide and polypeptide sequences of the signal transduction domains provided herein are known. In some embodiments, the polynucleotide encoding the signal transduction domain comprises 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%) the same polynucleotide sequence as a known nucleotide sequence. In some embodiments, the polypeptide sequence of the signal transduction domain comprises 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%) the same polypeptide sequence as a known polypeptide sequence.

In various implementations, mechanisms are needed to modulate (e.g., reduce) the activity of the antigen-binding system, for example, to minimize or reduce adverse events caused by the activity of the antigen-binding system. It may also be necessary to include an inducible "on" or "accelerator" switch in immune cells. Suitable techniques include the use of inducible cysteine-9 (US Patent Application 2011/0286980) or thymidine kinase before, after, or simultaneously with transduction of cells using the CAR construct of this disclosure. Other methods for introducing suicide genes and/or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques.

According to this disclosure, on-off or other types of control switching techniques are incorporated herein by reference. These techniques may include the use of dimerizing domains and activators that optionally dimerize such domains, such as those disclosed in Wu et al., Science 2014 350 (6258), utilizing the FKBP/Rapalog dimerizing system in certain cells, the contents of which are incorporated herein by reference in their entirety. Further dimerizing 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; 5,869,337; and 6,165,787, each of which is incorporated herein by reference. Other dimerization pairs may include cyclosporine-A/cyclophilic receptor, estrogen/estrogen receptor (optionally using tamoxifen, 4-hydroxytamoxifen, or endoxifen), glucocorticoid/glucocorticoid receptor, tetracycline/tetracycline receptor, and/or vitamin D/vitamin D receptor. Other 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 Nos. 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 system of this disclosure comprises a leader peptide (also referred to herein as a “signal peptide” or “lead sequence”). In some embodiments, the leader peptide comprises 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%) of the amino acid sequence MEWTWVFLFLLSVTAGVHS (SEQ ID NO:249), MALPVTALLLPLALLLHAARP (SEQ ID NO:250), or MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:295).

The components of a CAR can be swapped or "exchanged" with conventional biotechnological techniques to form equivalent components. For the purpose of providing only some non-limiting and partial examples, the CAR of this disclosure may comprise a combination of the binding motif, hinge, and co-stimulatory domain provided herein. In some instances, the CAR of this disclosure may comprise a leader sequence provided herein along with a combination of the binding motif, hinge, and co-stimulatory domain provided herein. In various embodiments, this disclosure provides a combination (e.g., adjacent fusion) of the binding motif according to any of SEQ ID NO:251-260 and the hinge according to any of SEQ ID NO:261-269, optionally further combined (e.g., adjacent fusion) with the 41BB co-stimulatory domain according to SEQ ID NO:270. Some non-limiting examples are provided in SEQ ID NO:271-290.

Bicistronic CARs may comprise a first CAR sequence and a second CAR sequence expressed as a single polypeptide containing a cleavable linker between the first and second CARs. An exemplary cleavable linker is furin-GSG-T2A (see, for example, Chng et al. MAbs. 2015 Mar-Apr; 7(2):403–412, the aspects of which regarding the cleavable linker are incorporated herein by reference; see also Guedan et al. Mol Ther Methods Clin Dev. 2019 Mar 15; 12:145–156, the aspects of which regarding the design of bicistronic CARs are incorporated herein by reference). For the purpose of providing only a non-limiting example of a bicistronic CAR structure, a bicistronic CAR may comprise (a) a first CAR comprising (i) a signal peptide (e.g., CSF2RA signal peptide); (ii) an anti-CD19 light chain variable domain; (iii) a linker (e.g., a G4S linker or multiple thereof); (iv) an anti-CD19 heavy chain variable domain; (v) a spacer region or hinge (e.g., a CD28T spacer region); (vi) a transmembrane domain (e.g., a CD28 transmembrane domain); (vii) a co-stimulatory domain (e.g., a CD28 co-stimulatory domain); (viii) a stimulatory domain (e.g., a CD3z stimulatory domain); and (b) a cleavable linker (e.g., furin G). (i) an SG-T2A linker; and (c) a second CAR comprising (i) a signal peptide (e.g., a CD8a signal peptide); (ii) an anti-CD20 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: 307-313); (iv) an anti-CD20 light chain variable domain of the present disclosure; (v) a spacer region or hinge (e.g., a CD8a spacer region); (vi) a transmembrane domain (e.g., a CD8 transmembrane domain); (vii) a co-stimulatory domain (e.g., a 41bb co-stimulatory domain); and (viii) a stimulatory domain (e.g., a CD3z stimulatory domain). Therefore, but not limited to, the exemplary anti-CD20/anti-CD19 bicistronic CAR may have or include the nucleotide and amino acid sequences shown in SEQ ID NO: 291 and 292.

A bispecific CAR may be a single polypeptide comprising a first binding motif of the present disclosure and a second binding motif as an anti-CD19 binding motif. In a non-limiting exemplary embodiment, a bispecific CAR may comprise (i) a leader (e.g., a CSF2RA signal peptide), (ii) an anti-CD20 light chain variable domain of the present disclosure; (iii) a linker; (iv) an anti-CD20 heavy chain variable domain; (v) a linker (e.g., a truncated linker); (vi) an anti-CD19 light chain variable domain; (vii) a linker; (viii) an anti-CD19 heavy chain variable domain; (ix) an extracellular domain (e.g., a CD28 T hinge or an IgG4 hinge); (x) a transmembrane domain (e.g., a CD28 transmembrane domain); (xi) an intracellular region (e.g., a CD28 intracellular co-stimulatory domain and/or a 41bb co-stimulatory domain); and a stimulatory domain (e.g., a CD3z stimulatory domain). Therefore, but not limited to, the exemplary anti-CD20/anti-CD19 bispecific CAR may have or include the nucleotide and amino acid sequences shown in SEQ ID NO:293-306.

Various CAR sequences, components, and/or frameworks are known, including but not limited to sequences of hinges, spacer regions, transmembrane domains, co-stimulatory domains, stimulatory domains, binding motifs, and their respective variants. CARs with desired binding and components or frameworks can be readily constructed if, for example, heavy chain variable domain sequences or CDR sequences and light chain variable domain sequences or CDR sequences are provided.

This disclosure particularly provides bispecific antibodies that bind to CD20 and a second target antigen, such as CD19. The bispecific antibody comprises an antibody having a first binding motif that binds to a first target antigen and a second binding motif that binds to a second target antigen. In some embodiments, the bispecific antibody comprises the anti-CD20 binding motif of this disclosure and the anti-CD19 binding motif of this disclosure. In some embodiments, the bispecific antibody comprises an anti-CD20 binding motif comprising the anti-CD20 heavy chain variable domain and the anti-CD20 light chain variable domain of this disclosure, and an anti-CD19 binding motif comprising the anti-CD19 heavy chain variable domain and the anti-CD19 light chain variable domain.

This disclosure includes conjugates in which the antibodies of this disclosure are associated with a therapeutic agent or a detectable moiety. In various embodiments, the therapeutic agent is the anticancer agent provided herein. In some embodiments, the provided conjugates comprise one or more detectable moieties, i.e., are “labeled” with one or more such moieties. In some such embodiments, the conjugates of this disclosure can be used for 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; fluorescent dyes; chemiluminescent agents (e.g., acridinium esters, stable dioxane, etc.); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (e.g., dyes, colloidal gold, etc.); biotin; digoxigenin; haptens; and proteins from which antiserum or monoclonal antibodies can be obtained.

This disclosure includes nucleic acids encoding anti-CD20 binding motifs and/or anti-CD19 binding motifs provided herein. This disclosure includes nucleic acids encoding antibodies provided herein, including but not limited to nucleic acids encoding binding motifs (e.g., anti-CD20 binding motifs and anti-CD19 binding motifs). This disclosure includes nucleic acids encoding 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 binding CD20 and CD19). The nucleic acid sequence of SEQ ID NO:2 includes 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 includes 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 includes and provides an exemplary nucleic acid sequence 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 an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 45 and 47-55. The nucleic acid sequence of SEQ ID NO: 57 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 56 and 58-66. The nucleic acid sequence of SEQ ID NO: 68 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 67 and 69-77. The nucleic acid sequence of SEQ ID NO: 79 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 78 and 80-88. The nucleic acid sequence of SEQ ID NO: 90 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 89 and 91-99. The nucleic acid sequence of SEQ ID NO: 101 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 100 and 102-110. The nucleic acid sequence of SEQ ID NO: 112 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 111 and 113-121. The nucleic acid sequence of SEQ ID NO: 123 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 122 and 124-132. The nucleic acid sequence of SEQ ID NO: 134 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 133 and 135-143. The nucleic acid sequence of SEQ ID NO: 145 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 144 and 146-154. The nucleic acid sequence of SEQ ID NO: 156 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 155 and 157-165. The nucleic acid sequence of SEQ ID NO: 167 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 166 and 168-176. The nucleic acid sequence of SEQ ID NO: 178 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 177 and 179-187. The nucleic acid sequence of SEQ ID NO: 189 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 188 and 190-198. The nucleic acid sequence of SEQ ID NO: 200 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 199 and 201-209. The nucleic acid sequence of SEQ ID NO: 211 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 210 and 212-220. This disclosure includes a nucleic acid encoding the anti-CD19 binding motif provided herein. The nucleic acid sequence of SEQ ID NO: 222 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 221 and 223-231. The nucleic acid sequence of SEQ ID NO: 233 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NO: 232 and 234-242.

This disclosure includes vectors comprising nucleic acids of this disclosure and/or polypeptides encoding the present disclosure. In various embodiments, this disclosure includes vectors comprising nucleic acids encoding anti-CD20 binding motifs and/or anti-CD19 binding motifs provided herein. In various embodiments, this disclosure includes vectors comprising nucleic acids encoding antibodies provided herein, which include, but are not limited to, nucleic acids encoding binding motif molecules (e.g., anti-CD20 binding motifs or anti-CD19 binding motifs). In various embodiments, this disclosure includes vectors comprising nucleic acids encoding one or more antigen-binding systems provided herein, which include, but are not limited to, nucleic acids encoding bicistronic or bispecific chimeric antigen receptors (e.g., bicistronic and bispecific chimeric antigen receptors binding CD20 and CD19).

Any vector may be suitable for this 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 adenovirus vector, a baculovirus vector, an EB virus vector, a lactovirus 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 largeTcDNA, 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-mCherry FP, pRetroX GFP T2ACre, pRXTN, pLncEXP, and pLXIN-Luc.

Recombinant expression vectors can 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, vectors can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors useful in the context of this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, bicistronic IRES vectors (e.g., from Clontech) are used to contain nucleic acids encoding an antigen-binding system and the inducible expression constructs described herein.

In some implementations, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alphaviruses, vaccines, adenoviruses, adeno-associated viruses, herpesviruses, and fowlpox virus vectors, and preferably have the ability to transform natural or engineered immune cells (e.g., T cells).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, 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 a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

Recombinant expression vectors can contain one or more biomarker genes, allowing selection of transformed or transfected hosts. Biomarker genes include antimicrobial resistance, such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts to provide the original nutrient. Suitable biomarker genes for recombinant expression vectors include, for example, neomycin/G418 resistance genes, puromycin resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

In the context of this disclosure, a useful vector may be a “naked” nucleic acid vector (i.e., a vector having little or no protein, sugar, and/or lipids encapsulating them) or a vector compounded with other molecules. Other molecules that can be suitably combined with a vector include, but are not limited to, viral capsids, cationic lipids, liposomes, polyamines, gold particles, and targeting motifs such as ligands, receptors, or antibodies targeting 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 techniques recognized in the art for introducing foreign nucleic acids (e.g., DNA) into cells, such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, liposome transfection, gene gun, nanoparticle-mediated delivery, or electroporation. Transduction involves viral delivery of a vector to cells, such as via vectors disclosed herein, including but not limited to retroviruses, lentiviruses, and AAVs.

This disclosure includes cells that contain, express, or are engineered (e.g., transformed or transduced) to contain or express at least one vector or nucleic acid of this disclosure. In some embodiments, the method includes transducing cells with a vector containing a polynucleotide encoding at least one antigen-binding system. This disclosure includes cells that contain or are transformed to contain a vector encoding at least one or more polypeptides of this disclosure. This disclosure includes cells that contain or are transformed to contain at least one vector encoding an anti-CD20 binding motif and/or an anti-CD19 binding motif provided herein. This disclosure includes cells that contain or are transformed to contain a vector encoding at least one antibody provided herein, which includes, but is not limited to, binding motif molecules (e.g., an anti-CD20 binding motif or an anti-CD19 binding motif). This disclosure includes cells that contain or are transformed to contain at least one vector encoding one or more antigen-binding systems provided herein, which include, but are not limited to, bicistronic or bispecific chimeric antigen receptors (e.g., bicistronic or bispecific chimeric antigen receptors that bind CD20 and CD19). In some implementations, cells are co-transfected or co-transduced using two vectors, each encoding a different CAR, which together form a bicistronic CAR. Transfection or transduction of cells with two different vectors (which together encode two different CARs that form a bicistronic CAR) can be performed simultaneously on a single cell population, simultaneously on two different cell populations, with each cell population transduced using only one of the two vectors, or independently on two different cell populations, with each cell population transduced using only one of the two vectors.

This disclosure includes cells comprising one or more polypeptides of this disclosure. This disclosure includes cells comprising (e.g., expressing) anti-CD20 binding motifs and/or anti-CD19 binding motifs provided herein. This disclosure includes cells comprising (e.g., expressing) antibodies provided herein, said antibodies comprising, but not limited to, binding motifs (e.g., anti-CD20 binding motifs or anti-CD19 binding motifs). This disclosure includes cells comprising (e.g., expressing) one or more antigen-binding systems provided herein, said antigen-binding systems comprising, but not limited to, bicistronic or bispecific chimeric antigen receptors (e.g., bicistronic and bispecific chimeric antigen receptors binding CD20 and CD19).

In other aspects, this document provides cells comprising the polynucleotides or vectors disclosed herein. In some embodiments, this disclosure relates to cells, such as in vitro cells, comprising a polynucleotide encoding a CAR or TCR comprising one or both of the scfvs disclosed herein. In other embodiments, this disclosure relates to cells, such as in vitro cells, 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 sequences or any combination thereof.

SEQ ID NO:232 (Anti-CD19 scFv light chain):

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

SEQ ID NO:221 (Anti-CD19 scFv heavy chain):

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

SEQ ID NO:56 (Anti-CD20 light chain):

DIQMTQSPSSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

SEQ ID NO:45 (Anti-CD20 Heavy Chain):

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSS

SEQ ID NO:56 (Anti-CD20scFv 2 light chain):

DIQMTQSPSSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

SEQ ID NO:155 (Anti-CD20scFv 2 heavy chain):

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSRYVWSWIRQPPGKGLEWIGEIDSSGKTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

SEQ ID NO:144 (Anti-CD20scFv 3 light chain):

DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK

SEQ ID NO:177 (Anti-CD20scFv 3-chain):

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYAWSWIRQPPGKGLEWIGEIDHRGFTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

SEQ ID NO:78 (Anti-CD20scFv 4 light chain):

DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK

SEQ ID NO:67 (Anti-CD20scFv 4-heavy chain):

QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS

For the polynucleotides, carriers, or polypeptides of this disclosure, any cell can be used as the host cell. In some embodiments, the cell can be a prokaryotic cell, fungal cell, yeast cell, or 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, such as Enterobacteriaceae (e.g., Escherichia coli), Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), and Serratia (e.g., Serratia depigmentans). Bacilli, such as *Bacillus subtilis* and *Bacillus licheniformis*; Pseudomonas, such as *Pseudomonas aeruginosa*; and Streptomyces. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are 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, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cells are T cells. In another embodiment, the immune cells are NK cells. In some implementations, the T cells are tumor-infiltrating lymphocytes (TILs), autologous T cells, engineered autologous T cells (eACT ^™ ), allogeneic T cells, xenogeneic T cells, or any combination thereof.

Chimeric antigen receptors (CARs or CAR-T) and engineered T-cell receptors (TCRs) can be readily inserted into and expressed by immune cells (e.g., T cells), thereby producing a binding agent. In some 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 with cancer or a tumor. In other embodiments, the donor subject is a human patient without cancer or a 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 disclosed herein can be obtained from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell populations in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL ^™ isolation and/or apheresis). In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in a suitable buffer or medium for further processing. In some embodiments, the cells are washed with PBS. As should be understood, the washing step can be used, for example, by using a semi-automatic non-reverse centrifuge (e.g., Cobe ^™ 2991 cell processor, BaxterCytoMate ^™ , etc.). In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other salt solutions with or without buffers. In some embodiments, unwanted components are removed from single samples. Further methods for isolating T cells for T-cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, which is incorporated herein by reference in its entirety.

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

In some embodiments, PBMCs are used directly for the genetic modification of immune cells using methods described herein (e.g., CAR or TCR). In some embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be sorted into naive T cells, memory T cells, and effector T cell subsets before or after genetic modification and/or expansion.

In some embodiments, CD8 ⁺ cells are further sorted into initial cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of the initial cell, central memory cell, and effector cell types of CD8 ⁺ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and they are granzyme B negative. In some embodiments, central memory T cells are CD8 ⁺ , CD45RO ⁺ , and CD62L ⁺ T cells. In some embodiments, effector T cells are CCR7, CD28, CD62L, and CD127 negative, and granzyme B and perforin positive. In some embodiments, CD4 ⁺ T cells are further sorted into subsets. For example, CD4 ⁺ T helper cells can be sorted into initial cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens.

In some embodiments, immune cells, such as T cells, are genetically modified after isolation using known methods, or the immune cells are activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In another embodiment, immune cells, such as T cells, are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR), and then activated and/or expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Patent Nos. 6,905,874; 6,867,041; and 6,797,514; and PCT Publication No. WO 2012/079000, the contents of which are incorporated herein by reference in their entirety. Generally, such methods involve contacting PBMCs or isolated T cells in a culture medium containing appropriate cytokines (e.g., IL-2) with stimulants and co-stimulants (e.g., anti-CD3 and anti-CD28 antibodies, typically adhered to beads or other surfaces). Anti-CD3 and anti-CD28 antibodies adhering to the same beads act as "surrogate" antigen-presenting cells (APCs). One example is a system for physiologically activating human T cells using a CD3/CD28 activator/stimulator. In other embodiments, methods described, for example, 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) are used to feed cells and activate and stimulate T cells for proliferation with appropriate antibodies and cytokines.

In all aspects of this disclosure, cells that contain, express, encode, or are transformed to encode a vector or polypeptide of this disclosure (e.g., the anti-CD20 binding motif and/or the anti-CD20/anti-CD19 antigen binding system of this disclosure) are binding agents. Binding agents or groups of binding agents may be used (e.g., as their active agents) as binding agents (compositions that can be used for therapeutic purposes, such as for cancer cells).

This disclosure further includes methods and processes for generating antibody agents as disclosed herein, such as by transforming (e.g., transducing) cells with a vector or nucleic acid of the present disclosure. In some embodiments, the methods or processes for generating antibody agents as disclosed herein include transforming (e.g., transducing) cells with a nucleic acid encoding at least one antigen-binding system provided herein (e.g., a nucleic acid present in a vector). Generally, the antibody agents described herein can be generated by immune cells, such as cells that can be used or are capable of being used for adoptive cell therapy. In some embodiments, the binder is generated by a cell type selected from the group consisting of: TILs, T cells, CD8 ⁺ cells, CD4 ⁺ cells, NK cells, gamma-delta T cells, regulatory T cells, or peripheral blood mononuclear cells. “Tumor-infiltrating lymphocytes” or TILs refer to white blood cells that have left the bloodstream and migrated into a tumor. Lymphocytes can be divided into three groups, which include B cells, T cells, and natural killer cells. “T cells” refer to CD3 ⁺ cells, which include CD4 ⁺ helper cells, CD8 ⁺ cytotoxic T cells, and gamma-delta T cells.

In some embodiments, the binder is produced by genetically modified (e.g., transformed) cells, such as immune cells having nucleic acids encoding the antigen-binding system described herein and/or expression constructs (e.g., (i) a first recombinant expression vector containing a nucleic acid encoding the antigen-binding system and a second recombinant expression vector containing an inducible expression construct; (ii) a single recombinant expression vector containing both the nucleic acid encoding the antigen-binding system and the inducible expression construct; or (iii) a recombinant expression vector containing a constitutive expression construct). The recombinant expression vector may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain nucleotide linkages that are naturally occurring or non-natural, or both types of linkages.

In some embodiments, the method includes transducing cells with a polynucleotide encoding an antigen-binding system as disclosed herein. In some embodiments, the method includes transducing cells with a vector containing a polynucleotide encoding an antigen-binding system.

In some embodiments, the donor T cells for T-cell therapy are obtained from a patient (e.g., for autologous T-cell therapy). In other embodiments, the donor T cells for T-cell therapy are obtained from a non-patient subject. In an exemplary aspect, the subject may undergo leukapheresis, in which leukocytes are collected, enriched, or depleted in vitro to select and/or isolate cells of interest, such as T cells. These T-cell isolates may be amplified and processed by methods to allow the introduction of one or more CAR constructs of this disclosure, thereby generating CAR T cells of this disclosure.

In some embodiments, immune cells are obtained from a subject and transformed, for example, transduced, using an inducible or constitutive expression construct as described herein, such as an expression vector comprising the inducible or constitutive expression construct described herein, to obtain a binding agent. Thus, in some embodiments, the binding agent comprises autologous cells from the same subject from whom the immune cells were obtained. In some embodiments, immune cells are obtained from a subject and transformed, for example, transduced, using an inducible or constitutive expression construct as described herein, such as an expression vector comprising the inducible or constitutive expression construct described herein, to obtain a binding agent for allogeneic transfer to another subject.

In some implementations, T cells are obtained from a donor subject. In some implementations, the donor subject is a patient with cancer or a tumor. In other implementations, the donor subject is a patient without cancer or a tumor.

The various compositions disclosed herein comprise engineered cell populations, which can be generated in any manner. In some embodiments, this disclosure provides human cell populations engineered to express antigen-binding systems 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 derived from a single human source, the single human source of which may be acceptable for administration of cultured populations in some embodiments. As disclosed herein, the binding agent may comprise any single cell or cell population, such as the engineered cell populations provided herein.

Other aspects of this disclosure relate to compositions comprising the polynucleotides, carriers, peptides, or in vitro cells 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 containing a TCD encoded by a polynucleotide of this disclosure. In yet another embodiment, the composition comprises T cells containing a CAR or TCR comprising one or both of the SCFVs disclosed herein.

In other embodiments, the composition is selected for use in parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. Preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In some embodiments, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8. In some embodiments, when considering parenteral administration, the composition is in a pyrogen-free, parenteral-acceptable aqueous solution comprising the composition described herein with or without an additional therapeutic agent in a pharmaceutically acceptable medium. In some embodiments, the medium for parenteral injection is sterile distilled water, wherein the composition described herein (with or without at least one additional therapeutic agent) is formulated into a suitably preserved sterile isotonic solution. In some embodiments, preparation involves a formulation having the desired molecule of a polymeric compound (e.g., polylactic acid or polyglycolic acid), beads, or liposomes that provides controlled or sustained release of the product, which is then delivered via depot injection. In some embodiments, an implantable drug delivery device is used to deliver the desired molecule.

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

In some embodiments, this disclosure provides pharmaceutical compositions comprising and/or delivering one or more cells as provided herein, such as binding agents encoding or expressing polypeptides provided herein, such as anti-CD20/anti-CD19 CARs (i.e., “binding pharmaceutical compositions”). Binding pharmaceutical compositions 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, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

The conjugate pharmaceutical compositions of this disclosure can 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 can be formulated for intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and/or subcutaneous administration routes. Preferably, the compositions are formulated for parenteral administration. Compositions suitable for parenteral administration can be aqueous or non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, bacteriostatic agents, and solutes, for example, that make the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The conjugate pharmaceutical compositions of this disclosure can be administered in a manner suitable for the disease to be treated (or prevented).

Sterile compositions for injection can be prepared using distilled water for injection as a medium according to routine pharmaceutical practice. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection, optionally in combination with suitable solubilizers, such as alcohols like ethanol, and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 ^™ , HCO-50, etc.

Non-limiting examples of oily liquids include sesame oil and soybean oil, and they can be combined with benzyl benzoate or benzyl alcohol to form a solubilizer. Other items that may be included are buffers such as phosphate buffers or sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injectable solution can be packaged in suitable ampoules.

In one embodiment, the binder pharmaceutical composition is substantially free of detectable levels of contaminants such as endotoxins, mycoplasma, replication-competent lentiviruses (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, combined human serum, bovine serum albumin, bovine serum, culture medium components, vector-packaged cell or plasmid components, bacteria, and fungi. In one embodiment, the bacteria are 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., binders, such as engineered T cells or engineered NK cells) can be activated and/or expanded from the binder pharmaceutical composition, and/or generate the binder pharmaceutical composition. In some embodiments, additional steps may be performed prior to administration to a subject. For example, the binder may be expanded in vitro after contacting (e.g., transduction or transfection) immune cells with the inducible or constitutive expression constructs described herein (e.g., expression vectors containing inducible or constitutive expression constructs). The in vitro expansion may be performed for 1 day or more prior to administration to a subject, e.g., 2 days or more, 3 days or more, 4 days or more, 6 days or more, or 8 days or more. In some embodiments, the in vitro expansion 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, the in vitro expansion may be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days prior to administration to a subject. A binder pharmaceutical composition comprising, for example, a binder (e.g., engineered T cells or engineered NK cells) can be formulated for administration at a desired dose, such as ^10⁴ to ^10⁹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ cells/kg body weight). Certain embodiments of this disclosure include methods of administering to a subject a pharmaceutical composition as described herein, such as, for example, a binder as described herein (e.g., engineered cell populations of this disclosure), a protein therapeutic agent as described herein, a composition comprising a binder, and/or a composition comprising a protein therapeutic agent, for example, to an amount that would effectively treat the subject when administered with an appropriate dosing regimen.

In some embodiments, the binder is autologous to the subject, and the subject may be immunologically naive, immune, diseased, or in another condition prior to the isolation of immune cells from the subject. In some embodiments, the binder may be stimulated with an antigen (e.g., a TCR antigen) during in vitro expansion. Antigen-stimulated expansion may optionally be supplemented with expansion under nonspecific stimulatory lymphocyte proliferation conditions, such as anti-CD3 antibodies, anti-Tac antibodies, anti-CD28 antibodies, or phytohemagglutinin (PHA). The expanded binder 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 binder is treated ex vivo with interleukin-2 (IL-2) before infusion into the cancer patient, and the cancer patient is treated with IL-2 after infusion. Additionally, in some embodiments, the cancer patient may undergo preparatory lymphodepletion—a temporary ablation of the immune system—before administration of the binder. The combination of IL-2 treatment and preparatory lymphodepletion can enhance the durability of the binder. In some embodiments, the binder is transduced or transfected with a nucleic acid encoding a cytokine, which may be engineered to provide constitutive, modulated, or time-controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that enhance T lymphocyte survival during the systolic phase, promoting the formation and survival of memory T lymphocytes.

In some embodiments, the conjugate is administered before, substantially simultaneously with, or after the administration of another therapeutic agent, such as a cancer treatment agent. The cancer treatment agent may be, for example, a chemotherapy agent, a biological agent, or radiation therapy. In some embodiments, the subject receiving the conjugate is not given treatment sufficient to cause immune cell depletion, such as lymphodepleting chemotherapy or radiation therapy.

In some implementations, the dose administered to the subject may vary depending on the implementation method, the composition used, the method of administration, and the site of treatment and the subject. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the therapeutically effective amount of a composition to be administered to a person or other subject to treat or prevent a medical condition. The precise amount of composition required for therapeutic effectiveness may depend on a variety of factors, such as the activity of the binder and the route of administration.

An appropriate number of binding cells may be administered to the subject. While a single binding cell described herein can expand and provide therapeutic benefit, in some embodiments, ^10² or more, such as ^10³ or more, ^10⁴ or more, ^10⁵ or more, or ^10⁸ or more binding cells are administered. In some embodiments, ^10¹² or fewer, such as ^10¹¹ or fewer, 10⁹ or fewer, ^10⁷ or fewer, or ^10⁵ or ^fewer binding cells described herein are administered to the subject. In some embodiments, ^10² – ^10⁵ , ^10⁴ – ^10⁷ , ^10³ – ^10⁹ , or ^10⁵ – ^10¹⁰ binding cells described herein are administered. A dose of the binding pharmaceutical composition, such as ^10⁴ to ^10⁹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ cells/kg body weight), may be administered. The conjugate pharmaceutical composition can be administered at doses of, for example, about ² x 10⁶ cells/kg, about ³ x ^10⁶ cells/kg, about ⁴ x 10⁶ cells/kg, about ⁵ x 10⁶ cells/kg, about ⁶ x 10⁶ cells/kg, about ⁷ x 10⁶ cells/kg, about ⁸ x 10⁶ cells/kg, about ⁹ x 10⁶ cells/kg, about ¹ x 10⁷ cells/kg, about ² x 10⁷ cells/kg, about ³ x 10⁷ cells/kg, about ⁴ x 10⁷ cells/kg, about ⁵ x 10⁷ cells/kg, about ⁶ x 10⁷ cells/kg, about ⁷ x 10⁷ cells/kg, about 8 x 10⁷ cells/kg, or about ⁹ x 10⁷ cells/kg.

The dosage of the binder described herein can be administered to mammals in a single dose or in a series of sub-dose over an appropriate period of time, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. The dosage unit containing an effective amount of the binder can be administered as a single daily dose, or the total daily dose can be administered as needed in two, three, four, or more sub-dose administrations per day.

A licensed physician may choose an appropriate route of administration. The route of administration may be parenteral, such as by injection, nasal administration, pulmonary administration, or percutaneous administration. Systemic or local administration may be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, the composition is selectively intended for parenteral delivery, inhalation, or delivery through the digestive tract, such as oral administration. The dosage and method of administration may vary depending on the subject's weight, age, condition, etc., and may be appropriately selected.

In various embodiments, the binders described herein may be incorporated into pharmaceutical compositions. Pharmaceutical compositions comprising the binders of this 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 cases, pharmaceutical compositions comprising the binders of this disclosure may be formulated to contain pharmaceutically acceptable carriers or excipients. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Compositions comprising the binders of this disclosure may contain pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

In various embodiments, distilled water for injection can be used as a medium, according to routine pharmaceutical practice, to formulate compositions containing binders as described herein, such as sterile formulations for injection. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection, optionally combined with suitable solubilizers, such as alcohols like ethanol, and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 ^™ , HCO-50, etc. As disclosed herein, pharmaceutical compositions containing binders can be in any form. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories.

The choice or use of any form may depend in part on the intended mode of administration and therapeutic application. For example, a composition containing a binder of the present disclosure intended for systemic or local delivery may be in the form of an injectable or infusionable solution. Thus, a composition containing a binder of the present disclosure may be formulated for administration via a parenteral route (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). Parenteral administration refers to a mode of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intrasacral, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracervical, and intrasternal injection and infusion.

The route of administration can be parenteral, such as by injection, nasal administration, pulmonary administration, or transdermal administration. It can be administered systemically or locally via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

In various embodiments, pharmaceutical compositions comprising the binder of this disclosure can be formulated as solutions, microemulsions, dispersants, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating a composition comprising the desired amount of the binder of this disclosure with one or more of the ingredients listed above into a suitable solvent, followed by sterilization by filtration, if necessary. Dispersants are typically prepared by incorporating a composition comprising the binder of this disclosure into a sterile medium containing a basic dispersion medium and any other desired ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying of the powder of the composition comprising the binder of this disclosure plus any additional desired ingredients from its previously sterile filtered solution (see below). Appropriate flowability of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. Extended absorption of injectable compositions containing the binders of this disclosure can be achieved by including agents that delay absorption, such as monostearate and gelatin, in the compositions containing the binders of this disclosure.

Pharmaceutical compositions containing the binders of this disclosure can 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, a pharmaceutical composition containing the binders of this disclosure can be formulated by suitably combining a therapeutic molecule with a pharmaceutically acceptable medium or carrier, such as sterile water and saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring excipient, diluent, carrier, preservative, or binder, and then mixing it in a unit dose form required by generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical formulation is an amount that provides a suitable dose within a specified range. Non-limiting examples of oily liquids include sesame oil and soybean oil, and it can 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, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injectable can be packaged in suitable ampoules.

In some embodiments, compositions containing the binder of this disclosure can be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In some embodiments, compositions containing the binder of this disclosure can be formulated for storage at 2-8°C (e.g., 4°C) 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, ten months, eleven months, one year, eleven and a half years, or two years). Thus, in some embodiments, compositions containing the binder of this disclosure are stably stored at 2-8°C (e.g., 4°C) for at least one year.

In some cases, pharmaceutical compositions comprising the binders of this disclosure may be formulated as solutions. In some embodiments, compositions comprising the binders of this disclosure may be formulated as buffer solutions, for example, at suitable concentrations and suitable for storage at 2-8°C (e.g., 4°C). Pharmaceutical compositions comprising the binders as described herein may be formulated in immunoliposome compositions. Liposomes having prolonged circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

In some embodiments, compositions comprising the binders of this disclosure may be formulated with a carrier that protects the composition from rapid release, such as controlled-release formulations comprising implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations are known. See, for example, J.R. Robinson (1978), "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

In some embodiments, compositions containing the binders of this disclosure can be formulated into compositions suitable for intrapulmonary administration (e.g., via inhaler or nebulizer) to mammals such as humans. Methods for formulating such compositions are known. Dry powder inhaler formulations and suitable systems for administering the formulations are also known. Lung administration can be oral and/or nasal. Examples of pharmaceutical devices for lung delivery include metered-dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, compositions containing the binders of this disclosure can be administered to the lungs of a subject via a dry powder inhaler. These inhalers are propellant-free devices that deliver a dispersible and stable dry powder formulation to the lungs. Dry powder inhalers are known and include, but are not limited to: (AstraZeneca; London, England) inhalers (Cambridge, Mass.); (GlaxoSmithKline; London, England); and ECLIPSE ^™ (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 the pulmonary administration of peptides such as insulin and growth hormone. In some embodiments, compositions comprising the binders of this disclosure can be administered intrapulmonaryly via a metered inhaler. These inhalers rely on a propellant to deliver discrete doses of molecules to the lungs. Additional devices and methods of intrapulmonary administration are set forth in, for example, U.S. Patent Application Publications 20050271660 and 20090110679, the respective disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, compositions comprising the binders of this disclosure may be formulated for delivery to the eye, for example, 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, tonics, antioxidants and stabilizers, nonionic wetting or clarifying agents, and thickeners. The formulations described herein may be applied topically to the eye of a subject requiring treatment (e.g., a subject with AMD) by conventional methods, such as in the form of drops containing one or more of the compositions, or by bathing the eye in a therapeutic solution.

In some embodiments, various devices for delivering drugs into the vitreous cavity of the eye can be adapted to administer compositions comprising the binders of this disclosure. For example, U.S. Publication No. 2002/0026176 describes a drug-containing plug that can be inserted through the sclera, thereby protruding into the vitreous cavity to deliver the drug 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 to continuously release a drug into the interior of the eye. U.S. Patent Nos. 5,773,019 and 6,001,386 each disclose an implantable drug delivery device that can be attached to the surface of the sclera of the eye. Other methods and devices for delivering therapeutic agents to the eye (e.g., via scleral patches and via contact lenses) are described, for example, in Ambati and Adamis (2002) Prog RetinEye 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 45:2342-2347; Kim et al. (2007) Ophthalmic Res 39:244-254; and PCT Publication No. WO 04/073551, the publication of which is incorporated herein by reference in its entirety.

In various implementations, subcutaneous administration can be accomplished by devices such as syringes, pre-filled syringes, autoinjectors (e.g., disposable or reusable), pen syringes, patch syringes, wearable syringes, dynamic syringe infusion pumps with subcutaneous infusion sets, or other devices used in combination with binder drugs for subcutaneous injection.

The injection system disclosed herein can employ a delivery pen as described in U.S. Patent No. 5,308,341. Pen-type devices are commonly used for self-delivery of insulin to patients with diabetes. Such devices may include at least one injection needle (e.g., a 31-gauge needle about 5 to 8 mm in length), which is typically pre-filled with one or more therapeutic unit doses of therapeutic solution and can be used to rapidly deliver the solution to the subject with minimal discomfort. A drug delivery pen includes a vial holder in which a vial of the therapeutic agent or other drug can be contained. The pen may be a fully mechanical device, or it may be combined with electronic circuitry to accurately set and/or indicate the dosage of the drug injected into the user. See, for example, U.S. Patent No. 6,192,891. In some embodiments, the needle of the pen-type device is disposable and the kit includes one or more disposable replacement needles. Pen-type devices suitable for delivering any currently characteristic composition containing a binder of the present disclosure are also described, for example, in U.S. Patent Nos. 6,277,099; 6,200,296; and 6,146,361, the respective disclosures 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 the MOLLY ^™ Precision Pen Injector (PPI) Device manufactured by Scandinavian Health Ltd.

In some embodiments, compositions comprising the binders of this disclosure can be delivered to a subject via topical application, independent of transport of the binder via the vascular system to its intended target tissue or site. For example, compositions comprising the binders of this disclosure can be delivered by injection or implantation of a composition comprising the binders of this disclosure, or by injection or implantation of a device containing a composition comprising the binders of this disclosure. In some embodiments, following topical application near a target tissue or site, a composition comprising the binders of this disclosure or one or more components thereof can diffuse to the intended target tissue or site, which is not the application site.

In some embodiments, compositions comprising the binders of this disclosure may be applied topically to joints, such as directly to the joint (e.g., in the joint space) or near the joint. Examples of intra-articular joints to which compositions comprising the binders of this disclosure may be applied topically include, for example, the hip, knee, elbow, wrist, sternoclavicular joint, temporomandibular joint, carpal joint, tarsal joint, ankle, and any other joint with an arthritic condition. Compositions comprising the binders of this disclosure may also be applied to bursae, such as, for example, the acromion, biceps radius, cubital radius, deltoid, subpatellar, ischium, and any other bursa.

In some embodiments, the compositions comprising the binders of this disclosure provided herein are available in unit dosage forms suitable for self-administration. Such unit dosage forms may be provided in containers, typically such as vials, cartridges, pre-filled syringes, or disposable pens. Dosing devices (such as those described in U.S. Patent No. 6,302,855) may also be used, for example, with the injection systems described herein.

The pharmaceutical solution may contain a therapeutically effective amount of a composition comprising a binder of the present disclosure. Such an effective amount may be readily determined, in part, based on the effect of the applied composition comprising a binder of the present disclosure, or the combined effect of the composition comprising a binder of the present disclosure and one or more other active agents (if more than one agent is used). The therapeutically effective amount of a composition comprising a binder of the present disclosure may also vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of the composition (and one or more other active ingredients) to elicit a desired response in the individual, such as improvement of at least one condition parameter, for example, improvement of at least one symptom of a complement-mediated condition. For example, a therapeutically effective amount of a composition comprising a binder of the present disclosure may inhibit (reduce its severity or eliminate its occurrence) and/or prevent the condition and/or any symptom of the condition. A therapeutically effective amount is also a therapeutically effective amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the composition comprising a binder of the present disclosure.

Compositions containing the binders of this disclosure may be administered as a fixed dose or at a dose of milligrams per kilogram (mg/kg). In some embodiments, the dose may also be selected to reduce or avoid the generation of antibodies against one or more antigen-binding molecules in the composition containing the binders of this disclosure or other host immune responses. While not intended to be limiting, exemplary doses of binders such as compositions containing the binders of this disclosure include, for example, 1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary doses of compositions containing the binders of this disclosure include, but are not limited to, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg.

Appropriate human doses of any composition containing a binder of this disclosure may be further evaluated, for example, in a Phase I dose-escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

In various embodiments, the pharmaceutical composition may comprise the nucleic acid of this disclosure, such as a carrier. Methods for formulating pharmaceutical compositions comprising the nucleic acid of this disclosure are known, for example, in the books in the series *Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs* (Dekker, N.Y.). For example, solutions or suspensions for parenteral, intradermal, or subcutaneous application may comprise the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and tonic agents, such as sodium chloride or glucose. The pH value may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in, for example, ampoules, disposable syringes, or vials made of glass or plastic for more than one dose.

Suitable injectable pharmaceutical compositions comprising the nucleic acids of this disclosure may contain a sterile aqueous solution or dispersant and a sterile powder for provisional preparation of a sterile injectable solution or dispersant. For intravenous administration, suitable carriers may comprise physiological saline, antibacterial water, Cremophor EL.™ (BASF, Parsippany, N.J.), or phosphate-buffered saline (PBS). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof.

Sterility, stability, viscosity, and other factors relevant to effective therapeutic use may be considered. For example, one method of maintaining flowability includes using coatings such as lecithin, maintaining the desired particle size, and using surfactants. In some cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. In some cases, prolonged absorption of injectable compositions containing the nucleic acids of this disclosure can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

Sterile injectable solutions can be prepared by incorporating one or more components, such as a combination of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), and/or by filtration sterilization. In some cases, dispersants are prepared by incorporating active molecules into a sterile medium containing a basic dispersing medium and an antibacterial or antifungal agent. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying from a previously sterile filtered solution.

Oral compositions containing the nucleic acids of this disclosure may contain an inert diluent or an edible carrier. For oral therapeutic administration, the nucleic acids of this disclosure may be combined with excipients and used in the form of, for example, tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions containing the nucleic acids of this disclosure may also be prepared using a fluid carrier used as a mouthwash. Pharmaceutically compatible binders and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following or molecules with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate or corn starch; lubricants such as magnesium stearate or glidants such as colloidal silica; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring.

In some implementations, nucleic acids can be administered by any method suitable for administering nucleic acid agents, such as DNA vaccines. These methods include, for example, gene guns, bioinjectors, 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 powdered vaccines, as disclosed in U.S. Patent No. 6,168,587. Furthermore, intranasal delivery is possible, as described in Hamajima et al., (1998) Clin. Immunol. Immunopathol. 88(2), 205-10. Liposomes (e.g., as described in U.S. Patent No. 6,472,375). In some cases, microencapsulation can be used. Additionally, biodegradable targeted microparticle delivery systems (e.g., as described in U.S. Patent No. 6,471,996) can be used.

Compositions containing the nucleic acids of this disclosure may include components such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, mixtures thereof, or any component used to contain in a therapeutic composition containing nucleic acids. Nucleic acid compositions may include, for example, saline solutions compatible with drug administration, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, phosphate-buffered saline solutions, water, and emulsions, such as oil/water or water/oil emulsions, as well as various types of wetting agents. Supplemental active molecules may also be incorporated into compositions containing the nucleic acids of this disclosure. Compositions containing the nucleic acids of this disclosure may include stabilizers and preservatives, as well as any carriers described herein, optionally with the additional condition that they are acceptable for in vivo use. For examples of other carriers, stabilizers and adjuvants, 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 manufacturing and using pharmaceutical compositions comprising nucleic acids of the present disclosure. Pharmaceutical compositions comprising nucleic acids of the present disclosure are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intracranial, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

Nucleic acid compositions may contain buffers or pH adjusters. Buffers may be salts prepared from organic acids or bases. Buffers disclosed herein contain 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, trimethylolaminomethane hydrochloride, and phosphate buffers. Additional carriers may contain polymeric excipients or additives such as polyvinylpyrrolidone, sucrose (a polymeric sugar), dextran (e.g., cyclodextrins, such as 2-hydroxypropyl-orthogonal-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as TWEEN and TWEEN), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

In some embodiments where the nucleic acid of this disclosure is a vector, this disclosure includes compositions for gene transduction and/or gene therapy, such as compositions containing viral particles, such as AAV particles, and/or retroviruses, such as lentiviral particles. As used with reference to viral titer, the term “genomic particle (gp)” or “genomic equivalent” refers to the number of viral particles containing a recombinant viral genome (e.g., a recombinant AAV DNA genome), regardless of infectivity or functionality. The number of genomic particles in a vector formulation can be measured. As used with 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, such as the number of recombinant AAV vector particles, which can be measured by an infection center assay (also known as a replication center assay). As used with reference to viral titer, 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 transgenic product.

In some embodiments, the composition comprises, for example, ^2x10⁶ to ^2x10¹² , ^2x10⁷ to ^2x10¹¹ , or ^2x10⁸ to ^2x10¹¹ DNA-containing viral particles per dose. In some embodiments, the concentration or titer of the carrier per unit dosage form is, for example, at least: (a) ¹ x 10¹² particles/mL, ² x 10¹² particles/mL, ³ x 10¹² particles/mL, ⁴ x 10¹² particles/mL, 5 x 10¹² particles/mL, ⁶ x ^10¹² particles/mL, 7 x 10¹² particles/mL, 8 x ^10¹² particles/mL, ⁹ x 10¹² particles/mL, ¹⁰ x 10¹² particles/mL, ¹⁵ x 10¹² particles/mL, ²⁰ x 10¹² particles/mL, ²⁵ x 10¹² particles/mL, or 50 x ^10¹² particles/mL; (b) ¹ x 10⁹ TU/mL, 2 x 10⁹ TU/mL, ³ x 10⁹ TU/mL, ⁴ x 10⁹ TU/mL, or ⁵⁰ x 10¹² particles/mL ^; TU/mL, 5x10 ⁹ TU/mL, 6x10 ⁹ TU/mL, 7x10 ⁹ TU/mL, 8x10 ⁹ TU/mL, 9x10 ⁹ TU/mL, 10x10 ⁹ TU/mL, 15x10 ⁹ TU/mL, 20x10 ⁹ TU/mL, 25 or 50x10 ⁹ TU/mL; or (c) 1x10 ¹⁰ IU/mL, 2x10 ¹⁰ IU/mL, 3x10 ¹⁰ IU/mL, 4x10 ¹⁰ IU/mL, 5x10 ¹⁰ IU/mL, 6x10 ¹⁰ IU/mL, 7x10 ¹⁰ IU/mL, 8x10 ¹⁰ IU/mL, 9x10 ¹⁰ IU/mL, 10x10 ¹⁰ IU/mL, 15x10 ¹⁰ IU/mL, ²⁰ x 10¹⁰ IU/mL, ²⁵ x 10¹⁰ IU/mL, or 50 x ^10¹⁰ IU/mL. Such embodiments do not limit the unit dose covered by this disclosure, nor do they limit the various measures of dose that can be used in conjunction with various compositions comprising the nucleic acids of this disclosure. For example, the dose, concentration, or amount of expressed particles can be measured in terms of vector genome/kilogram subject (Vg/Kg) or Vg/dose. Preferred methods for measuring and/or expressing particle dose, concentration, or amount may vary depending on various factors, such as the route of administration.

This disclosure provides techniques for simultaneously targeting CD20 and another antigen (e.g., CD19). In some embodiments, this disclosure provides techniques for initiating and/or modulating an immune response. In some embodiments, this disclosure provides techniques for treating cancer (e.g., cancers characterized by cells expressing CD20 on their surface).

This specification relates to the use of the binding pharmaceutical compositions provided herein for the treatment or prevention of cancer. Another aspect of this disclosure relates to methods for treating or preventing malignant tumors, methods comprising administering an effective amount of a binding pharmaceutical composition to a subject in need, for example, wherein cells comprise at least one antigen-binding system provided herein. The methods of this disclosure, including the administration of a pharmaceutically effective amount of a binding pharmaceutical composition of this disclosure, can be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumor from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the methods provided herein induce a complete response. In some embodiments, the methods provided herein induce a partial response. In some embodiments, the binding pharmaceutical composition is a cell provided herein, a cell comprising the cell provided herein, a cell comprising the cell provided herein as an active agent, or a cell comprising the cell provided herein as the sole active agent, such as a cell comprising or expressing at least one CAR of this disclosure. In some embodiments, the binder pharmaceutical composition comprises a bicistronic CAR system containing both anti-CD20 CAR and anti-CD19 CAR, or the binder pharmaceutical composition comprises the bispecific anti-C20/anti-CD19 CAR of this disclosure.

In various embodiments, this disclosure includes the use of the binding pharmaceutical compositions provided herein to induce or provide immunity against cancer in a subject. This disclosure further includes methods for preventing cancer in a subject by administering the binding pharmaceutical compositions provided herein to the subject. This disclosure further includes methods for inducing an immune response in a subject by administering the binding pharmaceutical compositions provided herein to the subject. In some embodiments, the binding pharmaceutical composition is a cell provided herein, a cell containing the cells provided herein, a cell containing the cells provided herein as an active agent, or a cell containing the cells provided herein as the sole active agent, such as a cell containing or expressing at least one CAR of this disclosure. In some embodiments, the binding pharmaceutical composition comprises a bicistronic CAR system comprising an anti-CD20 CAR and an anti-CD19 CAR, or the binding pharmaceutical composition comprises a bispecific anti-C20/anti-CD19 CAR of this disclosure.

In some embodiments, a method of treating a subject with cancer in need includes administering to the subject the polynucleotide, carrier, antibody, or antigen-binding system disclosed herein. In one embodiment, the method includes administering a polynucleotide encoding an antigen-binding system or antibody (e.g., an antigen-binding system). In another embodiment, the method includes administering a carrier containing a polynucleotide encoding an antigen-binding system or antibody. In yet another embodiment, the method includes administering an antigen-binding system or antibody to the subject.

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

Another aspect of this disclosure relates to a method for inducing immunity against a tumor, comprising administering to a subject an effective amount of cells comprising the polynucleotides, vectors, or CARs or TCRs described herein. In one embodiment, the method comprises administering to a subject an effective amount of cells comprising a polynucleotide encoding a CAR or TCR disclosed herein. In another embodiment, the method comprises administering to a subject an effective amount of cells comprising a vector comprising a polynucleotide encoding a CAR or TCR disclosed herein. In yet another embodiment, the method comprises administering to a subject an effective amount of cells comprising a CAR or TCR encoded by a polynucleotide disclosed herein.

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

Another aspect of this disclosure relates to a method for treating or preventing malignant tumors, the method comprising administering an effective amount of at least one immune cell to a subject in need, 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 this disclosure relates to a method of treating cancer in a subject with this need, comprising administering to the subject the polynucleotides, vectors, CARs or TCRs, cells, or compositions disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding a CAR or TCR. In another embodiment, the method comprises administering a vector containing a polynucleotide encoding a CAR or TCR. In yet another embodiment, the method comprises administering a CAR or TCR encoded by a polynucleotide disclosed herein. In yet another embodiment, the method comprises administering cells containing a polynucleotide encoding a CAR or TCR or a vector containing a polynucleotide encoding a CAR or TCR.

In some embodiments, the method of treating cancer in a subject with this need includes T-cell therapy. In one embodiment, the T-cell therapy of this disclosure is engineered autologous cell therapy (eACT ^™ ). According to this embodiment, the method may include collecting blood cells from a patient. The isolated blood cells (e.g., T cells) may then be engineered to express the CAR or TCR of this disclosure. In a specific embodiment, CAR T cells or TCR T cells are administered to the patient. In some embodiments, CAR T cells or TCR T cells treat a tumor or cancer in a patient. In one embodiment, CAR T cells or TCR T cells shrink 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 non-patient subject.

T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells may be at least about ^10⁴ cells, at least about ^10⁵ cells, at least about ^10⁶ cells, at least about ^10⁷ cells, at least about ^10⁸ cells, at least about ^10⁹ cells, or at least about ^10¹⁰ cells. In another embodiment, a therapeutically effective amount of T cells is about ^10⁴ cells, about ^10⁵ cells, about ^10⁶ cells, about ^10⁷ cells, or about ^10⁸ cells. In one specific implementation, the therapeutically effective dose of CAR T cells or TCR T cells is approximately ² x 10⁶ cells/kg, approximately ³ x 10⁶ cells/kg, approximately ⁴ x 10⁶ cells/kg, approximately ⁵ x 10⁶ cells/kg, approximately ⁶ x 10⁶ cells/kg, approximately ⁷ x 10⁶ cells/kg, approximately ⁸ x 10⁶ cells/kg, approximately ⁹ x 10⁶ cells/kg, approximately ¹ x 10⁷ cells/kg, approximately ² x 10⁷ cells/kg, approximately ³ x 10⁷ cells/kg, approximately ⁴ x 10⁷ cells/kg, approximately ⁵ x 10⁷ cells/kg, approximately ⁶ x 10⁷ cells/kg, approximately ⁷ x 10⁷ cells/kg, approximately ⁸ x 10⁷ cells/kg, or approximately ⁹ x 10⁷ cells/kg.

The methods disclosed herein can be used to treat cancer in a subject, 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 patient remission, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods elicit a partial response.

In some embodiments, the cancer includes cells expressing CD19, such as cells expressing CD19 on their cell surface. In some embodiments, the cancer includes cells expressing CD20, such as cells expressing CD20 on their cell surface. In some embodiments, the cancer includes cells that independently express both CD19 and CD20, such as on their cell surface.

Treatable cancers include non-vascularized, substantially non-vascularized, or vascularized tumors. Cancers can also include solid tumors or non-solid tumors. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is a cancer of white blood cells. In other embodiments, the cancer is a cancer of plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or myeloma. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute lymphoblastic leukemia (ALL) and hemophagocytic lymphohistiocytosis (HLH), B-cell prolymphoblastic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic or acute granulomatous disease, chronic... Acute or non-acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), hairy cell leukemia, hemophagocytic syndrome (macrophage activation syndrome (MAS), Hodgkin's disease, large cell granuloma, leukocyte adhesion defect, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplastic syndromes (M... DS), myeloid diseases including but not limited to acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), plasmacytic proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell tumor, plasmacytoma (e.g., plasmacytic cachexia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (Crow-Fukase syndrome; Takatsuki disease; 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 lymphoblastic leukemia (“TALL”), T-cell lymphoma, transformed follicular lymphoma, or Warschow's macroglobulinemia, or a combination thereof. In one embodiment, the cancer is myeloma. In one 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 situations, the method of treating cancer using the binding drug compositions provided herein is autologous cell therapy. In various situations, the method of treating cancer using the binding drug compositions provided herein is allogeneic cell therapy.

Some methods of using the pharmaceutical compositions using binders provided herein include collecting blood cells from a subject. The isolated blood cells from the subject (e.g., T cells) can then be engineered to express, for example, an antigen-binding system disclosed herein. In some embodiments, the binder is administered to the subject. In some embodiments, the binder treats the subject's cancer. In one embodiment, the binder reduces the size of a tumor.

In various embodiments, the cell therapies provided herein for use in this disclosure can be administered to a subject during treatment, said treatment further comprising the administration of one or more additional therapeutic agents or therapies not provided herein. In some embodiments, this disclosure provides combination therapies for the treatment of cancer, said treatment comprising the administration of an anticancer agent to a subject receiving and/or requiring a combination of the agents provided herein.

In some embodiments, the conjugates provided herein may be administered to subjects who have previously received, are planned to receive, or are in the course of a treatment regimen that includes additional anticancer therapies. In various embodiments, additional agents or therapies administered in combination with the conjugates provided herein, as described herein, may be administered concurrently with the conjugates provided herein, on the same day as the conjugates provided herein, or in the same week as the conjugates provided herein. In various embodiments, additional agents or therapies administered in combination with the conjugates provided herein, as described herein, may be administered such that the administration of the conjugates provided herein and the administration of the additional agents and therapies are spaced one or more hours before or after the administration, one or more days before or after the administration of the conjugates provided herein, one or more weeks before or after the administration, or one or more months before or after the administration. In various embodiments, the frequency of administration of one or more additional agents may be the same as, similar to, or different from the frequency of administration of the conjugates provided herein.

The reagents or therapies used in combination with the binders provided herein may be administered together with the binders provided herein as a single therapeutic composition or dose, as a separate composition, or in a manner different in timing from the administration of the binders provided herein. When the binders provided herein are used in combination with other reagents, the binders provided herein may be formulated together with the other reagents or separately from the other reagent formulations.

In some embodiments, the method further comprises administering a chemotherapeutic agent. In some embodiments, the selected chemotherapeutic agent is a lymphoreductive (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens and associated 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 for conditioning patients requiring T-cell therapy, comprising administering to the patient a prescribed beneficial dose of cyclophosphamide (200 mg/ ^m² /day–2000 mg/ ^m² /day) and a prescribed dose of fludarabine (20 mg/ ^m² /day–900 mg/ ^m² /day). One such dosing regimen involves treating a patient by administering to the patient approximately 500 mg/ ^m² /day of cyclophosphamide and approximately 60 mg/ ^m² /day of fludarabine daily for 3 days, followed by administration of a therapeutically effective amount of engineered T cells. In other embodiments, the antigen-binding molecule, the transduced (or otherwise engineered) cells (e.g., CAR or TCR), and the chemotherapeutic agent are each administered in an amount effective in treating the subject's disease or condition.

In some embodiments, the composition comprising immune effector cells expressing CAR and/or TCR as disclosed herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ^™ ); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine resume; and nitrogen mustards. Mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil. Mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin C, and calicheamicin. Cin), carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin D, daunorubicin, detorubicin, 6-diazol-5-oxo-L-leucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid (Acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, and triamcinolone acetonide. Trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, and 5-FU; androgen derivatives, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid. acid); amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; podophyllinic acid acid); phenamet; pirarubicin; losoxantrone; ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2”-trichlorotriethylamine;urethan;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactalol;piperobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL ^™ , Bristol-Myers Squibb) and docetaxel (Rhone-Poulenc) Rorer); chlorambucil; gemcitabine; thioguanine; mecaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitosis Mycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoic acid derivatives, such as Targretin ^™ (bexarotene), Panretin ^™ (alitretinoin); ONTAK ^™ (denileukindiftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing substances. In some embodiments, the composition comprising CAR and/or TCR-expressing immune effector cells may be administered in combination with an antihormonal agent that modulates or inhibits the effect of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, and cyproheptadine. Keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing substances. In appropriate cases, combinations of chemotherapeutic agents may also be administered, including but not limited to CHOP, namely cyclophosphamide doxorubicin (hydroxydoxorubicin), vincristine, and prednisone.

In some embodiments, the chemotherapy agent is administered concurrently with or within one week following the administration of engineered cells or nucleic acids. In other embodiments, the chemotherapy agent is administered 1 to 4 weeks, 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 the administration of engineered cells or nucleic acids. In some embodiments, the chemotherapy agent is administered at least 1 month before the administration of cells or nucleic acids. In some embodiments, the method further comprises administering two or more chemotherapy agents.

Several other therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful other therapeutic agents include PD-1 inhibitors such as nivolumab, pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche). Other therapeutic agents suitable for use in combination with this disclosure include, but are not limited to, ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, trastuzumab emtansine, imatinib, cetuximab, panitumumab, catutoxumab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, and gemtuzumab. Erlotinib, Gefitinib, Vandetanib, Afatinib, Lapatinib, Neratinib, Axitinib, Masitinib, Pazopanib, Sunitinib, Sorafenib, Toceranib, Lestaurtinib, Axitinib, Cedilanib, Lenvatinib, Nintedanib, Pazopanib, Regofenib Sorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib nib), selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukindiftitox, mTOR inhibitors such as everolimus and simeolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as palbociclib.

In another embodiment, a composition comprising immune cells containing CARs and/or TCRs is administered in conjunction with an anti-inflammatory agent. The anti-inflammatory agent or drug may include, but is not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Examples of NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate. Examples of analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Examples of glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules targeting cell surface markers (such as CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept, adalimumab, and infliximab), and monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.

In some embodiments, the compositions described herein are administered in combination with cytokines. As used herein, "cytokine" means a protein released by one cell population that acts as an intercellular mediator to another cell. Examples of cytokines are lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental prolactin; Müller's duct-inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF) such as NGF-beta; platelet-derived growth factor; and transforming growth factor (TGF) such as TGF-alpha. Phyta- and TGF-beta; insulin-like growth factor-I and-II; erythropoietin (EPO); bone-inducing 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 (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and 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 of natural origin or derived from recombinant cell cultures, as well as bioactive equivalents of naturally occurring sequence cytokines. In various embodiments, the binders provided herein for use in this disclosure can be administered to a subject during treatment, the treatment further comprising the 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), nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate mofetil. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol, and propoxyphene citrate. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules targeting cell surface markers (such as CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept, adalimumab, and infliximab), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and molecules in recombinant forms. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (orally (auranofin) and intramuscularly), and minocycline.

In various embodiments, the binding agent provided herein for use in this disclosure can be administered to a subject during a treatment process, said treatment process further comprising the administration of CHOP. CHOP comprises: (C) cyclophosphamide, an alkylating agent that disrupts DNA by binding to DNA and causing the formation of cross-links; (H) hydroxydaunorubicin (also known as doxorubicin or doxorubicin), an intercalating agent that disrupts DNA by inserting itself between DNA bases; (O) vincristine, which inhibits cell replication by binding to the protein tubulin; and (P) prednisone or prednisolone, which is a corticosteroid.

Other exemplary embodiments. This disclosure includes, but is not limited to, the following exemplary embodiments:

Implementation Scheme 1. An isolated polynucleotide encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR) comprising (i) an antigen-binding molecule, (ii) a co-stimulatory domain, and (iii) an activation domain, wherein the co-stimulatory domain comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen-binding molecule, the antigen-binding molecule being substantially composed of or consisting of: (i) an amino acid sequence that is 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 any of the constructs described in SEQ ID NO: 232, 221, 56, 45, 155, 144, 177, 78, and 67.

Implementation Scheme 2. The polynucleotide of Implementation Scheme 1, wherein the transmembrane domain is a transmembrane domain of any of the following: 4-1BB/CD137, the alpha chain of the T cell receptor, the beta chain of the T cell receptor, CD3epsilon, CD4, CD5, CD8alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of the T cell receptor, or any combination thereof.

Implementation Scheme 3. The polynucleotide of Implementation Scheme 1 or 2, wherein the intracellular domain comprises the signal transduction regions of the following: 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 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49 D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD11, CD11, CD11, CD11, CD11, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, immunoglobulins Similar proteins, inducible T cell co-stimulatory molecules (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITG1, KIRDS2, LAT, LFA-1, LFA-1, ligands that specifically bind to CD83, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a/CD18), MHC class I molecules, NKG2C, NKG2 D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or combinations thereof.

Implementation Scheme 4. The polynucleotides of Implementation Schemes 1 to 3, wherein at least one of the antigen-binding molecules specifically binds to an antigen selected from the group consisting 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, ER BB2, 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 gp120, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1 or VEGFR2, or combinations thereof.

Implementation Scheme 5. A polynucleotide of any one of Implementation Schemes 1 to 4, wherein the polynucleotide encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO 232, 221, 56, 45, 155, 144, 177, 78 and 67.

Implementation Scheme 6. A polypeptide encoded by any one of the polynucleotides in Implementation Schemes 1 to 5.

Implementation Scheme 7. A polypeptide comprising the amino acid sequence shown in SEQ ID NO. 232, 221, 56, 45, 155, 144, 177, 78 and 67.

Implementation Scheme 8. A vector containing any one of the polynucleotides in Implementation Schemes 1 to 5.

Implementation Scheme 9. The vector of Implementation Scheme 7, wherein the vector is an adenovirus vector, an adenovirus-associated vector, a DNA vector, a lentiviral vector, a plasmid, a retroviral vector, or an RNA vector, or any combination thereof.

Implementation Scheme 10. A cell comprising a polynucleotide of any one of Implementation Schemes 1 to 5, a polypeptide of claim 6 or 7, a carrier of claim 8 or 9, or any combination thereof.

Implementation Scheme 11. A composition comprising a polynucleotide of any one of Implementation Schemes 1 to 5, a polypeptide of claim 6 or 7, a carrier of claim 8 or 9, a cell of claim 10, or any combination thereof.

Implementation Scheme 12. A method for preparing cells, said cells comprising a polynucleotide of any one of claims 1 to 5, a polypeptide of claim 6 or 7, a carrier of claim 8 or 9, or any combination thereof.

Implementation Scheme 13. A method for inducing immunity against a tumor, comprising administering to a subject an effective amount of any of the polynucleotides of claims 1 to 5, the polypeptide of implementation Scheme 6 or 7, the carrier of implementation Scheme 8 or 9, the cell of implementation Scheme 10, the composition of implementation Scheme 11, or any combination thereof.

Implementation Scheme 14. The use of any polynucleotide of Implementation Scheme 1 to 5, the polypeptide of Implementation Scheme 6 or 7, the carrier of Implementation Scheme 8 or 9, the cell of Implementation Scheme 11, or the composition of Implementation Scheme 12 for the manufacture of a medicament for the treatment of cancer in a subject in need.

Implementation Plan 15. Use of Implementation Plan 15, wherein the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphoblastic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid 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 disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplastic syndromes, and myelodysplastic syndromes. The following are considered asymptomatic myeloma, non-Hodgkin's lymphoma (NHL), plasmacytic proliferative disorders (including asymptomatic myeloma (accumulated multiple myeloma or indolent myeloma)), plasmablastic lymphoma, plasmacytoid dendritic cell tumor, plasmacytoma (including plasmacytic cachexia; 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 lymphoblastic leukemia (“TALL”), T-cell lymphoma, transformed follicular lymphoma, or Warschow's macroglobulinemia, or a combination thereof.

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and separately indicated to be incorporated herein by reference. However, the citation of references herein should not be construed as an admission that such reference is prior art to this invention. Where any definition or terminology provided in any cited reference differs from the terminology and discussion provided herein, the terminology and definitions of this invention shall prevail. The contents of all references cited throughout this application are expressly incorporated herein by reference.

Example

Example 1

This embodiment provides exemplary generated anti-CD20 heavy chain variable domains and light chain variable domains, and combinations thereof. Exemplary CDR sequences of the anti-CD20 heavy chain variable domain and the anti-CD20 light chain variable domain, and exemplary combinations of the resulting HCDR and LCDR, are also provided in Tables 4-13 below. Tables 4-13 contain exemplary nucleic acid sequences encoding exemplary variable domains (and therefore also provide exemplary nucleic acid sequences encoding identified CDRs of exemplary variable domains).

To determine the cell binding of the exemplary heavy chain variable domain and light chain variable domain of this disclosure, the cell binding of purified IgG at a concentration of 10 nM was characterized by flow cytometry. Antibodies were incubated with CHO-S cells overexpressing CD20, Raji and Namalwa CD20+ cell lines; and with EoL-1 and CHO-S CD20– cell lines. FITC-LC was used to detect IgG. The binding ratio of each antibody relative to the negative control was calculated. Exemplary cell binding of selected anti-CD20 antibodies is provided in Table 15.

Table 15

Example 2

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

The bispecific CAR of this embodiment comprises the following domains: a first binding motif, a second binding motif, a hinge, a transmembrane domain, and an intracellular domain comprising a co-stimulatory domain and an activation domain. In this embodiment, each bispecific CAR includes a first binding motif comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain and light chain variable domains are the heavy chain and light chain variable domains of a single set of exemplary antibody sequences from Example 1 (i.e., both are from the same table in Tables 4-13, corresponding to Ab1-Ab10). Therefore, the binding motif can be identified by referring to the source antibody or the source table, and refers to a binding motif having the heavy chain and light chain variable domains of the source antibody as listed in the corresponding table. In this embodiment, each bispecific CAR includes a second binding motif comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain and light chain variable domains are the heavy chain and light chain variable domains of antibody Ab11 (i.e., SEQ ID NO: 221 and 232). The bispecific CAR of this embodiment includes a 28T (CD28) domain comprising a hinge domain and a transmembrane domain. The bispecific CAR of this embodiment also includes a CD28 co-stimulatory domain. Furthermore, the bispecific CAR of this embodiment includes a CD3z activation domain.

This embodiment includes four bicistronic CARs, designated Bic-2, Bic-8, Bic-9, and Bic-14. Each of the four bicistronic CARs includes a first CAR construct containing an anti-CD20 binding motif, the anti-CD20 binding motif comprising a heavy chain variable domain and a light chain variable domain pair from Example 1, as shown in Table 16 below, and a second CAR construct containing an anti-CD19 binding motif.

Table 16: Anti-CD20 binding motif sequences of bicistronic CARs

CAR Combining 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

CD4 ⁺ and CD8 ⁺ T cells were isolated by positive selection from single-collected material from healthy donors and used to generate anti-CD20 monovalent or anti-CD20/anti-CD19 bicistronic CAR T cell products. T cells were activated with conjugated anti-CD3 and soluble CD28 antibodies and transduced using a lentiviral vector encoding the CAR construct. As a control, non-transduced (NTD) T cells were generated in parallel from the same donor T cells. On harvest day (days 8–10 of manufacturing), the CAR T cell products were stained and analyzed by flow cytometry to assess transduction efficiency and were used for co-culture assays. The transduction efficiency of T cells using vectors encoding monovalent CARs and vectors encoding bicistronic CARs was monitored.

To determine the T-cell transduction efficiency of vectors encoding monovalent CARs, CAR-T products were stained with a panel of antibodies (anti-CD3, anti-CD4, anti-CD8, and anti-adaptor antibodies) in the presence of a fixable viability dye, and the percentage of surviving CAR-positive cells was assessed by flow cytometry (see WO/US2017/041534, which relates to anti-adaptor antibodies and is incorporated herein by reference). Anti-adaptor antibodies are antibodies that bind to the linker between the heavy and light chains of the anti-CD20 CAR-binding motif and are used to measure transduction efficiency. Controls included non-transduced cells (NTD), cells transduced with a retrovirus containing a control anti-CD19 binding agent, and cells transduced with a control anti-CD20 binding agent (Ab12 binding motif).

To determine the T-cell transduction efficiency of vectors encoding bicistronic CARs, CAR-T products were stained with a panel of antibodies (containing anti-CD3, anti-CD4, anti-CD8, anti-idiotype, and anti-adaptor antibodies) in the presence of a fixable viability dye, and the percentage of surviving CAR-positive cells was assessed by flow cytometry. The anti-idiotype antibody binds to the Ab11 anti-CD19 binding motif. Therefore, the anti-idiotype antibody binds to the anti-CD19 CAR. It was used to measure the transduction efficiency of the anti-CD19 CAR. The anti-adaptor antibody was used to measure the transduction efficiency of the anti-CD20 CAR. Controls included non-transduced cells (NTD), cells transduced with a retrovirus containing a control anti-CD19 binder, and cells transduced with control anti-CD20/anti-CD19 bispecific CARs (Ab13/Ab14 bispecific; Ab11/Ab12 bispecific).

Table 17A: Transduction efficiency of anti-CD20 monovalent CAR

Combining motifs # 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-CD20/anti-CD19 bicistronic CAR

Example 4

This embodiment provides, in particular, an exemplary method for co-culturing CAR-T cells with cells expressing CAR-T target antigens. To facilitate tracking of the cultured T cells, CAR-T cells are labeled with CellTrace ^™ Violet (CTV) reagent according to the manufacturer's instructions and then washed with R-10% medium. To facilitate tracking of cells expressing CAR-T target antigens (“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. Furthermore, Nalm6 and Raji cells that do not express CD19 or CD20 (knockout cells, or KO) were prepared. These CD19KO and CD20KO cells were selected from Nalm6 and Raji parental cell clones and expressed either CD20 but not CD19, or CD19 but not CD20, respectively. CD19KO and CD20KO strains were generated and used as controls to functionally evaluate antigen binding of each CAR in cells expressing bicistronic anti-CD20/anti-CD19 CARs.

Target cells expressing luciferase were plated together with CTV-labeled CAR-T cells at varying ratios in R-10% medium (day 0 of co-culture). This ratio can be referred to as the effector (CAR-T) cell to target cell ratio (effector:target or E:T). To plate cells at the desired ratio, CAR-T cells were serially diluted 2 to 3 times while maintaining the target cell count at 25,000 cells per well. The co-cultures were incubated at 37°C for 16 hours (h) or 4 days and functionally evaluated as described below.

Example 5

In this embodiment, as described in Example 4, T cells were co-cultured with target cells. T cell-mediated cytotoxicity was measured as a function of the reduction in the target luciferase signal in the 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 0.14 mg/mL, and the plates were incubated in the dark at 37°C for 10 minutes. The luminescence signal was immediately read in a VarioSkan ^™ LUX or Flash multimode microplate reader. T cell-mediated cytotoxicity was calculated as follows: %cytotoxicity = [1 – (luciferase signal of sample of interest / target control alone)] * 100.

Controls included non-transduced (NTD) T cells (i.e. T cells that do not express CAR) as negative controls, cells transduced with a retrovirus containing a control anti-CD19 binder, cells transduced with a control anti-CD20 binder, and cells transduced with a control anti-CD20/anti-CD19 bispecific CAR (Ab13/Ab14 bispecific; Ab15/Ab16 bispecific).

Table 18: Percentage of anti-CD20 CAR-T cytotoxicity in co-cultures after 4 days (Nalm6 wild-type cells)

Table 19: Percentage of anti-CD20 CAR-T cytotoxicity in co-cultures after 4 days (Raji wild-type cells)

Table 20: Percentage of cytotoxicity of anti-CD20/anti-CD19 bicistronic CAR-T cells in co-cultures after 4 days (Nalm6 wild-type cells)

Table 21: Percentage of cytotoxicity of anti-CD20/anti-CD19 bicistronic CAR-T cells in co-cultures after 4 days (Raji wild-type cells)

Table 22: Percentage of cytotoxicity of anti-CD20/anti-CD19 bicistronic CAR-T cells in co-cultures after 4 days (Nalm6CD19KO cells)

Table 23: Percentage of cytotoxicity of anti-CD20/anti-CD19 bicistronic CAR-T cells in co-cultures after 4 days (Nalm6CD20KO cells)

Table 24: Percentage of cytotoxicity of anti-CD20/anti-CD19 bicistronic CAR-T cells in co-cultures after 4 days (RajiCD19KO cells)

Table 25: Percentage of cytotoxicity of anti-CD20/anti-CD19 bicistronic CAR-T cells in co-cultures after 4 days (RajiCD20KO cells)

Example 6

In this embodiment, as described in Example 4, T cells were co-cultured with target cells. After 16 hours of co-culture, the supernatant was collected and cytokine levels were analyzed using the Meso Scale Discovery V-PLEX Pro-inflammatory Group 1-Person Kit according to the manufacturer's instructions. The levels of antigen-conjugated interferon gamma (IFN-γ), IL-2, tumor necrosis factor alpha (TNF-α), and IL-10 secreted from the supernatant of T cell products plated at a 1:1 E:T ratio with target cells expressing the antigen were analyzed. All samples were diluted to the detection range. The level of each cytokine was reported in pg/mL, and the lower and upper limits of quantitation for each assay were also reported.

The controls included non-transduced (NTD) T cells (i.e. T cells that do not express CAR) as negative controls, cells transduced with a retrovirus containing a control anti-CD19 binder, cells transduced with a control anti-CD20 binder, cells transduced with a control bispecific antigen binding system (Ab13/Ab14 bispecific), and cells transduced with a bispecific antigen binding system (Ab15/Ab16 bispecific).

Table 26: Cytokine production (pg/mL) in 16-hour co-culture of anti-CD20 CAR-T cells and Nalm6 wild-type cells

Table 27: Cytokine production (pg/mL) in 16-hour co-culture of anti-CD20 CAR-T cells and Raji wild-type cells

Table 28: Cytokine production (pg/mL) in 16-hour co-culture of anti-CD20/anti-CD19 bicistronic CAR-T cells and Nalm6 wild-type cells.

Table 29: Cytokine production (pg/mL) in 16-hour co-culture of anti-CD20/anti-CD19 bicistronic CAR-T cells and Raji wild-type cells.

Table 30: Cytokine production (pg/mL) in 16-hour co-culture of anti-CD20/anti-CD19 bicistronic CAR-T cells and Nalm6 CD19KO cells.

Table 31: Cytokine production (pg/mL) in 16-hour co-culture of anti-CD20/anti-CD19 bicistronic CAR-T cells and 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 set of antibody fluorophores to identify T cells (CD3, CD4, CD8) and 4-1BB (an activation marker), and analyzed by flow cytometry. The immobilizable viability dyes allowed for the analysis of viable cells. Events were systematically gated on viable cells (viability dye negative), lymphocytes (using forward scatter [FSC] regions and side scatter [SSC] regions), single cells (using FSC regions and FSC height maps), and then on T cells (CD3+). The activation level of T cells (i.e., the percentage of 4-1BB ⁺ cells) was then analyzed; 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 do not express CAR) as negative controls, cells transduced with a retrovirus containing a control anti-CD19 binder, cells transduced with a control anti-CD20 binder, 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-CD20 CAR-T cells in 16-hour co-culture with Nalm6 wild-type and Raji wild-type cells

Table 33: Activation of anti-CD20/anti-CD19 bicistronic CAR-T cells in 16-hour co-culture 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 the 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 with antigen-expressing target cells at a 3:1 E:T ratio were harvested. Viable T cells were identified by staining with a set of antibody fluorophores (CD3, CD4, CD8) in the presence of a viability-fixing dye and analyzed by flow cytometry. The percentage of proliferating cells and the mean fluorescence intensity (MFI) of the CTV signal were reported. A decrease in the MFI of CTV was proportional to the number of cell division rounds experienced by the product (i.e., the lower the CTV MFI, the greater the proliferation experienced by the cells).

The controls included non-transduced (NTD) T cells (i.e. T cells that do not express CAR) as negative controls, cells transduced with a retrovirus containing a control anti-CD19 binder, cells transduced with a control anti-CD20 binder, cells transduced with a control bispecific antigen binding system (Ab13/Ab14 bispecific), and cells transduced with an Ab11/Ab12 bispecific antigen binding system.

Table 34: Fluorescence of anti-CD20 CAR-T cells in four-day co-cultures with Nalm6 wild-type and Raji wild-type cells

Table 34: Percentage of anti-CD20 CAR-T cells proliferated in four-day co-cultures with Nalm6 wild-type and Raji wild-type cells

Nalm6 WT Raji WT Combining motifs # 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: Fluorescence of anti-CD20/anti-CD19 bicistron CAR-T cells in four-day co-cultures with Nalm6 wild-type, Nalm6 CD19KO, Raji wild-type, and Raji CD19KO cells

Table 36: Percentage of anti-CD20/anti-CD19 bicistronic CAR-T cells proliferated in four-day co-cultures with Nalm6 wild-type, Nalm6 CD19KO, Raji wild-type, and Raji CD19KO cells

Example 9

This embodiment provides data related to the hinge of the anti-CD20 antigen binding system. The CAR-T cells in this embodiment contain a monovalent anti-CD20 CAR. This embodiment tests the CAR-T construct against a Raji human B-cell lymphoma model in NSG mice that disseminates luciferase. CAR-T persistence in blood samples was monitored weekly. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. Whole blood was drawn and analyzed 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.

The CAR-T cells used in this study are shown in Table 37 below, where each of the CAR-T cell types in Table 37 was administered to six mice. Table 37 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. All CAR-T cells were administered intravenously at a dose of 2E+07 cells/mL (QDx1). All mice also received a dose of 36 μg of human IL-2 ((Q12Hx2)QDx3). Human IL-2 promotes the persistence and survival of CAR-T cells. The CAR T cell constructs in groups 4, 5, and 7-10 of Table 37 contain a 41BB co-stimulatory domain. The CAR T cells in group 6 of Table 37 contain a CD28 co-stimulatory domain.

Table 37:

The study mice tolerated all CAR-T cell therapies well. Within each group, numerous endpoints 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. Results are shown in Table 38 below; note that data reflect monitoring performed by terminating the experiment on day 55.

Table 38

Example 10

This embodiment provides data related to the antitumor efficacy of CAR-T cells containing bicistronic CARs, which include anti-CD19 CARs and anti-CD20 CARs, with the anti-CD20 CARs containing various anti-CD20 binding motifs and/or hinges.

Bicistronic anti-CD20/anti-CD19 CAR-T cells were tested against disseminated luciferase-expressing Raji CD19KO B-cell lymphoma in NSG mice. CAR-T persistence in blood samples was monitored weekly. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. Whole blood was drawn and analyzed 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 used in this study, as shown in rows 5-14 of Table 39 below, were bicistronic CAR-T cells containing anti-CD20 CAR and anti-CD19 CAR (Ab11 CAR containing a 28T (CD28) hinge and CD28 co-stimulatory domain) as shown in Table 39. Table 39 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. CAR-T cells were administered to six mice (except for four mice that 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 cells/mL (rows 8, 10, 12, and 14) (QDx1). All mice also received 36 μg of human IL-2 ((Q12Hx2)QDx3).

Table 39

Many 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%). Results are shown in Table 40 below; note that data reflect monitoring conducted by terminating the experiment on day 55.

The mice in the study tolerated all CAR-T cell therapies well. All tested CAR-T cell therapies elicited an antitumor response. All treatment regimens resulted in a median tumor growth delay of >30.2 days and an increase in intratumor lifespan (ILS) of >226.7%. Except for treatment with non-transduced (NTD) cells (24%), all treatment regimens resulted in a median %T/C of 0% at day 19.

Table 40

Example 11

This embodiment provides data related to the antitumor efficacy of CAR-T cells containing a bispecific CAR comprising an anti-CD19 binding motif and an anti-CD20 binding motif. Bispecific anti-CD20/anti-CD19 CAR-T cells were tested against disseminated Raji B-cell lymphoma tumors expressing luciferase in NSG mice. CAR-T persistence in blood samples was monitored weekly. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. Whole blood was drawn and analyzed 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 rows 4-8 of Table 41 below, the CAR-T cells used in this study are bispecific CAR-T cells containing both an anti-CD19 binding motif and an anti-CD20 binding motif, with the anti-CD19 binding motif being Ab11. Table 41 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. CAR-T cells were administered at specific doses to multiple mice, as shown in Table 41. All CAR-T cells were administered intravenously (QDx1). All mice also received a dose of 36 μg of human IL-2 ((Q12Hx2)QDx3).

Table 41

Many 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 life expectancy (ILS%). Results are shown in Table 42 below. Antitumor efficacy was observed.

Table 42

Example 12

This embodiment provides data on the antitumor efficacy of (i) CAR-T cells comprising bicistronic CARs containing both anti-CD19 and anti-CD20 CARs; and (ii) bispecific CARs comprising both anti-CD19 and anti-CD20 binding motifs. Bicistronic and bispecific anti-CD20/anti-CD19 CAR-T cells were tested against disseminated Raji B-cell lymphoma tumors expressing luciferase in NSG mice. CAR-T persistence in blood samples was monitored weekly. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. Whole blood was drawn and analyzed 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 used in this study, as shown in Table 43 below, are bicistronic or bispecific CAR-T cells containing an anti-CD20 binding motif and an anti-CD19 binding motif (Ab11). Table 43 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. 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 (QDx1). All mice also received an intraperitoneal dose of 36 μg of human IL-2 ((Q12Hx2)QDx3).

Table 43

1 PBS control 2 CAR-T cell control (non-transduced T cells) (60.0E+06 cells) 3 CAR-T cells containing anti-CD19 control CAR (2.6E+06 cells) 4 CAR-T cells containing anti-CD19 CAR with a linker according to SEQ ID NO:247 5 CAR-T cells containing anti-CD19 CARs with G4S linkers 6 Including CAR-T cells containing bicistronic CARs with binding motif #2. 7 Including CAR-T cells containing bicistronic CARs that bind motif #9. 8 Including CAR-T cells containing bicistronic CARs with binding motif #14 9 Including CAR-T cells containing a bispecific CAR that binds motif #2 10 Including CAR-T cells containing a bispecific CAR that binds motif #9 11 Including CAR-T cells containing a bispecific CAR that binds motif #14 12 CAR-T cells containing control bispecific anti-CD20/anti-CD19 CAR 13 CAR-T cells containing control bispecific anti-CD20/anti-CD19 CAR

Many 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%). Results are shown in Table 44 below. Note that the data reflect monitoring conducted by terminating the experiment on day 48.

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

Table 44

Example 13

This embodiment provides data related to the antitumor efficacy of CAR-T cells containing a monovalent anti-CD20 CAR. Anti-CD20 CAR-T cells were tested against disseminated Raji B-cell lymphoma tumors (CD19WT) expressing luciferase in NSG mice. CAR-T persistence was monitored weekly in blood samples. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. BLI was performed at days 5, 12, 21, 28, 33, 40, and 47. The study ended on day 55. The CAR-T cells used in this study are shown in Table 45 below, administered at the doses and number of mice shown in Table 45. Table 45 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. All CAR-T cells were administered intravenously (QDx1). All mice received a dose of 36 μg of human IL-2 ((Q12Hx2)QDx3).

Table 45

Many 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 life expectancy (ILS%). The results are shown in Table 46 below.

Table 46

Example 14

This embodiment provides data on the antitumor efficacy of CAR-T cells including bicistronic CARs containing anti-CD19 and anti-CD20 CARs, and data on the antitumor efficacy of CAR-T cells containing monovalent anti-CD20 CARs. CAR-T cells were tested against Raji B-cell lymphoma tumors expressing luciferase in NSG mice. CAR-T persistence was monitored weekly in blood samples. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. BLI was performed at days 5, 12, 19, 26, 33, and 40.

The CAR-T cells used in this study are shown in Table 47 below. Table 47 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. Groups 1-10 contain only control and monospecific CAR-T cells, while groups 11-16 contain bicistronic CAR-T cells. Bicistronic CAR-T cells contain anti-CD20 CAR and anti-CD19 CAR (Ab11 CAR) as shown in Table 39. Each condition shown in Table 47 contains 6 mice. All CAR-T cells were administered intravenously at a specified dose (QDx1). All mice also received an intraperitoneal dose of 36 μg of human IL-2 ((Q12Hx2)QDx3).

Table 47

Many 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%). Results are shown in Table 48 below. The study mice tolerated all CAR-T cell therapies well.

Table 48

Example 15

This embodiment provides data on the antitumor efficacy of CAR-T cells including bicistronic CARs containing anti-CD19 and anti-CD20 CARs, as well as data on the antitumor efficacy of CAR-T cells containing monovalent anti-CD20 CARs. CAR-T cells were tested in a NALM6 human acute lymphoblastic leukemia model expressing luciferase in NSG mice. CAR-T persistence in blood samples was monitored. Response was assessed based on bioluminescence imaging (BLI) and survival endpoints. Whole blood was drawn and analyzed 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.

The CAR-T cells used in this study are shown in Table 49 below. Table 49 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. Groups 1-8 contain only control and monospecific CAR-T cells, while groups 9-12 contain bicistronic CAR-T cells. Bicistronic CAR-T cells contain anti-CD20 CAR and anti-CD19 CAR (Ab11 CAR) as shown in Table 49. Each condition shown in Table 49 includes 6 mice. All CAR-T cells were administered intravenously at the specified dose (QDx1).

Table 49

Many 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%). Results are shown in Table 50 below. The study mice tolerated all CAR-T cell therapies.

Table 50

Example 16

This embodiment provides data related to the sequences used as CAR components. Four anti-CD20 binding motifs were tested in combination with four hinges (CD8 hinge (8k), truncated CD28 hinge (28T), truncated CD28 hinge with G4S linker; and IgG4 hinge (I4)). All bispecific and bicistronic CARs contain the Ab11 anti-CD19 binding motif. CD4 ⁺ and CD8 ⁺ T cells were isolated by positive selection from single-harvest material from healthy donors and used to generate anti-CD20 monovalent or anti-CD20/anti-CD19 bicistronic CAR T cell products. T cells were activated with bound anti-CD3 and soluble CD28 antibodies and transduced with a lentiviral vector encoding the CAR construct. As a control, non-transduced (NTD) T cells were generated in parallel from the same donor T cells. On harvest day (days 8–10 of manufacturing), CAR T cell products were stained and analyzed by flow cytometry to assess transduction efficiency and used for co-culture assays. The transduction efficiency of T cells was monitored using vectors encoding monovalent CARs and vectors encoding bicistronic CARs.

To determine the T-cell transduction efficiency of vectors encoding monovalent CARs, CAR-T products were stained with a panel of antibodies (anti-CD3, anti-CD4, anti-CD8, and anti-adaptor) in the presence of a fixable viability dye, and the percentage of surviving CAR-positive cells was assessed by flow cytometry. The anti-adaptor antibody, which binds to the adapter between the heavy and light chains of the anti-CD20 CAR-binding motif, was used to measure transduction efficiency. Controls included non-transduced cells (NTD), cells transduced with a retrovirus containing a control anti-CD19 binding agent, and cells transduced with a control anti-CD20 binding agent (Ab12 binding motif).

To determine the T-cell transduction efficiency of vectors encoding bicistronic CARs, CAR-T products were stained with a panel of antibodies (containing anti-CD3, anti-CD4, anti-CD8, anti-idiotype, and anti-adaptor) in the presence of a fixable viability dye, and the percentage of surviving CAR-T positive cells was assessed by flow cytometry. The anti-idiotype antibody binds to the Ab11 anti-CD19 binding motif. Therefore, the anti-idiotype antibody binds to the anti-CD19 CAR. It was used to measure the transduction efficiency of the anti-CD19 CAR. The anti-adaptor antibody was used to measure the transduction efficiency of the anti-CD20 CAR. Controls included non-transduced cells (NTD), cells transduced with a retrovirus containing a control anti-CD19 binder, and cells transduced with control anti-CD20/anti-CD19 bispecific CARs (Ab13/Ab14 bispecific; Ab11/Ab12 bispecific).

To facilitate tracking of T cells in culture, CAR-T cells were labeled with CellTrace ^™ Violet (CTV) reagent according to the manufacturer's instructions and then washed with R-10% medium. To facilitate tracking of cells expressing CAR-T target antigens (“target cells”), the target cells were engineered to express luciferase. Luciferase-expressing target cells included Nalm6 and Raji, both of which express both CD19 and CD20 antigens. Additionally, Nalm6 and Raji cells that did not express CD19 or CD20 (knockout cells, or KO) were prepared. These CD19KO and CD20KO cells were selected from Nalm6 and Raji parental cell clones and expressed either CD20 but not CD19, or CD19 but not CD20, respectively. CD19KO and CD20KO lines were generated and used as controls to functionally evaluate antigen binding for each CAR in cells expressing bicistronic anti-CD20/anti-CD19 CARs.

Target cells expressing luciferase were plated together with CTV-labeled CAR-T cells at varying ratios in R-10% medium (day 0 of co-culture). This ratio can be referred to as the effector (CAR-T) cell to target cell ratio (effector:target or E:T). To plate cells at the desired ratio, CAR-T cells were serially diluted 2 to 3 times while maintaining the target cell count at 25,000 cells per well. The co-cultures were incubated at 37°C for 16 hours (h) or 4 days, and functional assessments were performed as described below.

T cell-mediated cytotoxicity was measured as a function of the reduction in target luciferase signal in co-culture wells compared to the signal emitted by target cells plated alone. On day 4 after co-culture began, D-luciferin substrate was added to the co-culture wells at a final concentration of 0.14 mg/mL, and the plates were incubated in the dark at 37°C for 10 minutes. The luminescence signal was immediately read in a VarioSkan ^™ LUX or Flash multimode microplate reader. T cell-mediated cytotoxicity was calculated as follows: %cytotoxicity = [1 – (luciferase signal of sample of interest / target control alone)] * 100.

Cytokine production was measured after 16 hours of co-culture. The supernatant was then collected and cytokine levels were analyzed using the Meso Scale Discovery V-PLEX Pro-inflammatory Group 1-Person Kit, following the manufacturer's instructions. The levels of antigen-conjugated interferon gamma (IFN-γ), IL-2, tumor necrosis factor alpha (TNF-α), and IL-10 secreted from the supernatant of T cell products co-cultured with antigen-expressing target cells at a 1:1 E:T ratio were analyzed. All samples were diluted to the detection range. Levels of each cytokine were reported in pg/mL, along with the lower and upper limits of quantitation for each assay.

To determine T cell activation after 16 hours of co-culture, T cell products were harvested and plated with antigen-positive target cells at a specific E:T ratio. A set of antibody fluorophores was used to identify T cells (CD3, CD4, CD8) and 4-1BB (an activation marker), and the results were analyzed by flow cytometry. A viability-fixable dye allowed for analysis of viable cells. Events were systematically gated on live cells (viability-negative), lymphocytes (using forward scatter [FSC] and side scatter [SSC] region mapping), single cells (using FSC region and FSC height mapping), and then on T cells (CD3+). T cell activation levels (i.e., the percentage of 4-1BB ⁺ cells) were then analyzed; the 4-1BB gating threshold was set based on the expression level of NTD control T cells.

The proliferative capacity of T cell products 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 co-culture, T cell products plated with antigen-expressing target cells at a 3:1 E:T ratio were harvested. Viable T cells were identified by staining with a set of antibody fluorophores (CD3, CD4, CD8) in the presence of immobilizable viability dyes and analyzed by flow cytometry. The percentage of proliferating cells and the mean fluorescence intensity (MFI) of the CTV signal were reported. Decreased MFI of CTV was proportional to the number of cell division rounds experienced by the product.

The CAR-T cells used in this embodiment comprise the CARs identified in Table 51 below. Table 51 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. Transduction efficiency is shown in Table 52. Cytotoxicity on day 4 in Nalm6 (CD19+; low CD20), Nalm6CD19KO (CD19-; CD20+), Raji (CD19+, CD20+), and Raji CD19KO (CD19-, CD20+) is shown in Tables 53A-53D. Cytokine production 14 hours after co-culturing in Nalm6 CD19KO target cells at a 1:3E:T ratio is shown in Tables 54A-54D, measured in pg/mL (2 replicates/condition). T cell activation, measured by upregulation of 41BB (percentage of 41BB+ viable T cells) 16 hours after co-culturing, is shown in Tables 55A-D (2 replicates per condition). The proliferation of T cells on day 4 after co-culture is shown in Table 56 (percentage of CD3+ proliferation, 2 replicates for each condition).

Table 51

Table 52

Group 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 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 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 twenty one twenty two twenty three twenty four %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

Table 56

Example 17

Hinge comparisons were performed on anti-CD20 monovalent CARs. The data provided in this embodiment were obtained using the methods described in the above embodiments. The CAR-T cells used in this embodiment comprise the CARs shown in Table 57 below. Table 57 provides the experimental conditions or groups mentioned in subsequent tables of this embodiment. This embodiment utilizes four anti-CD20 binding motifs, shown in this embodiment as binding motif A, binding motif B, binding motif C, and binding motif D. The hinges tested comprised truncated CD28 hinges (28T), CD8 hinges (8k), and IgG4 hinges (I4).

Transduction efficiency is shown in Table 58. Cytotoxicity on day 4 in Raji (CD19+, high CD20), Raji CD20KO (CD19+, CD20-), Namalwa (CD19+, low CD20), and Nalm6 (CD19+, low CD20) is shown in Tables 59 and 60. Cytokine production 14 hours after co-culture at a 1:1 E:T ratio is shown in Tables 61A-D. T cell activation measured by upregulation of 41BB at 16 hours after co-culture in E:T 1:1 or 1:4 conditions is shown in Tables 62 and 63, respectively. T cell proliferation 4 days after co-culture in E:T 1:1 or 1:4 conditions is shown in Tables 64 and 65, respectively.

Table 57

Table 58

Group 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

Table 59

Table 60

Table 61A

Table 61B

Table 61C

Table 61D

Table 62

Table 63

Table 64

Table 65

Example 18

Table 66: Exemplary Combining Motif Sequences

Example 19

Table 67: Exemplary Hinge Sequences

Example 20

Table 68: Exemplary nucleotide sequences encoding binding motifs, hinges, and 41BB co-stimulatory domains

Example 21

Table 69: Exemplary anti-CD20/anti-CD19 bicistronic CAR nucleotide and amino acid sequences shown in SEQ ID NO: 291 and 292.

Example 22

Table 70: Exemplary anti-CD20/anti-CD19 bispecific CARs and their components

Example 23

Table 71: Exemplary Connectors

While multiple embodiments have been described, it is apparent that this disclosure and examples may provide other embodiments utilizing or covered by the compositions and methods described herein. Therefore, it should 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 illustrated by example.

sequence list

<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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Gly Gly Ser Phe Ser Gly Tyr Tyr

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

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

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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

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Arg Ala Ser Gln Ser Ile Ser Trp Leu Ala

1 5 10

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<220>

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Arg Ala Ser Gln Ser Ile Ser Trp Leu Ala

1 5 10

<210> 17

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Asp Ala Ser Ser Leu Glu Ser

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<211> 7

<212> PRT

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Asp Ala Ser Ser Leu Glu Ser

1 5

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<211> 7

<212> PRT

<213> Artificial Sequence

<220>

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Asp Ala Ser Ser Leu Glu Ser

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<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

polypeptide

<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 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

Polynucleotides

<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

polypeptide

<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

Polynucleotides

<400> 35

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gaggccacc 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

polypeptide

<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

Polynucleotides

<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 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 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 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

polypeptide

<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

Polynucleotides

<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 acctttcac 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

polypeptide

<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

Polynucleotides

<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

polypeptide

<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

Polynucleotides

<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

polypeptide

<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

Polynucleotides

<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

polypeptide

<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

Polynucleotides

<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 acctttcac 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

polypeptide

<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 Thr Val Thr Val Ser Ser

115 120

<210> 112

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

Polynucleotides

<400> 112

cagatcacct tgaaggagtc tggtcctacg ctggtgaaac ccacacagac cctcacgctg 60

acctgcacct tctctgggtt ctcactcgac actgaaggag tgggtgtggg ctggatccgt 120

cagcccccag gaaaggcct 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

polypeptide

<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

Polynucleotides

<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 acctatcac 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

polypeptide

<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 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

Polynucleotides

<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 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 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 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

polypeptide

<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

Polynucleotides

<400> 145

gacatcgtgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gaggccacc 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

polypeptide

<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 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

Polynucleotides

<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 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 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 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

polypeptide

<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

Polynucleotides

<400> 167

gacatcgtgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gaggccacc 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

polypeptide

<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 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

Polynucleotides

<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 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 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 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

polypeptide

<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

Polynucleotides

<400> 189

gacatcgtgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gaggccacc 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

polypeptide

<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 Tyr Ile Thr Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr 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

Polynucleotides

<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

polypeptide

<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

Polynucleotides

<400> 211

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gaggccacc 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

polypeptide

<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 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 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

Polynucleotides

<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 acatattattac 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 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 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

polypeptide

<400> 232

Asp Ile Gln Met Thr Gln Thr Thr Ser 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

Polynucleotides

<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

polypeptide

<400> 243

Asp Ile Gln Met Thr Gln Thr Thr Ser 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

Polynucleotides

<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 caagccccgga 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

Polynucleotides

<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 gttatactgccg 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 gaggaccaa ggttgagatc aag 813

<210> 252

<211> 834

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

Polynucleotides

<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 gttatactgccg 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

Polynucleotides

<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

Polynucleotides

<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

Polynucleotides

<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 gggttatactat ttctgtggac 300

acaagcaaaa atcagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcggggatgg 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

Polynucleotides

<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 acaggttac 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

Polynucleotides

<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 gttatactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttat 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 aggagagcg 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

Polynucleotides

<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 gttatactgccg 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 aggagagcg 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

Polynucleotides

<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 gttatactgccg 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 aggagagcg 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

Polynucleotides

<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 gttatactgccg 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

Polynucleotides

<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

Polynucleotides

<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

Polynucleotides

<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

Polynucleotides

<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

Polynucleotides

<400> 265

ggaggaggag gatctggtgg aggaggttct ctggacaatg agaaatcgaa tgggacaatc 60

atccatgtga agggggaagca 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

Polynucleotides

<400> 266

ggaggaggag gatctggtgg aggaggttct ctggacaatg aaaagagcaa tggcacaatc 60

atccatgtga agggggaagca 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

Polynucleotides

<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

Polynucleotides

<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 agaccaccccc 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

Polynucleotides

<400> 269

gagagaaagt gttgtgttga gtgtcctcct tgtcctcct gccctgctcc cgagttactt 60

ggcggacctt cagtgttcct gttccccccc aagcccaagg atactctcat gatcagccgg 120

accccccgagg 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 tgttatccc tgctggtcac tgtagctttc 780

atcatatttt gggtg 795

<210> 270

<211> 494

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

Polynucleotides

<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

Polynucleotides

<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 aggggcctgta 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

Polynucleotides

<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 gggttatactat ttctgtggac 300

acaagcaaaa atcagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcggggatgg 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 aggggcctgta 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

Polynucleotides

<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 acaggttac 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

Polynucleotides

<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 gttatactgccg 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 agaaacag 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 gagacccga gatgggcggc 1380

aaacctagaa gaaagaaccc ccaggagggc ctgtataacg agctccagaa ggacaagatg 1440

gccgaggcct acagcgagat cggcatgaag ggcgaaagaa gaagaggcaa gggccacgac 1500

ggcctctacc agggcttaag cacagctaca aaggacacct acgacgcct gcacatgcag 1560

gccctgcccc ctagatgatt aattaaatcg at 1592

<210> 275

<211> 1604

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

Polynucleotides

<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 gttatactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttat 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 aggagagcg 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 tgcagaccc acaggaggaa 1200

gacggctgca gctgtagatt ccccgaggaa gagagggcg 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

Polynucleotides

<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 gttatactgccg 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 aggagagcg 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 tgcagaccc acaggaggaa 1200

gacggctgca gctgtagatt ccccgaggaa gagagggcg 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

Polynucleotides

<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 gttatactgccg 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 aggagagcg 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 tgcagaccc acaggaggaa 1200

gacggctgca gctgtagatt ccccgaggaa gagagggcg 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

Polynucleotides

<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 gttatactgccg 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 gaggaccaa 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

Polynucleotides

<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

Polynucleotides

<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 gttatactgccg 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

accccccgctc 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 acaaggagg aagacggctg cagctgtaga 1200

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1260

gcccctgcct accagcaagg agaatcaa ctgtacaacg agctgaacct gggcagacgg 1320

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1380

agaagaaaga accccagga 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

Polynucleotides

<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 attattct 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

Polynucleotides

<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 gggttatactat ttctgtggac 300

acaagcaaaa atcagttcag cctgaagctg agctctgtga ctgccgccga cacagctgtg 360

tactactgtg ccagagagac agactactcc agcggggatgg 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 attattct 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

Polynucleotides

<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 acaggttac 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 agaatcaa 1200

ctgtacaacg agctgaacct gggcagacgg gaggaatacg atgtgctgga caagaggaga 1260

ggcagagacc ccgagatggg cggcaaacct agaagaaaga accccagga 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

Polynucleotides

<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 gttatactgccg 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

Polynucleotides

<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 gttatactgccg ccgacacagc tgtgtactat 360

tgcgccagag ttagatacga cagcagcgac agctattact acagctatga ctacggcatg 420

gatgtgtggg ggcagggcac caccgttat 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 aggagagcg 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 acaaggagg aagacggctg cagctgtaga 1140

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1200

gcccctgcct accagcaagg agaatcaa ctgtacaacg agctgaacct gggcagacgg 1260

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1320

agaagaaaga accccagga 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

Polynucleotides

<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 gttatactgccg 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 aggagagcg 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 acaaggagg aagacggctg cagctgtaga 1140

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1200

gcccctgcct accagcaagg agaatcaa ctgtacaacg agctgaacct gggcagacgg 1260

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1320

agaagaaaga accccagga 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

Polynucleotides

<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 gttatactgccg 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 aggagagcg 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 acaaggagg aagacggctg cagctgtaga 1140

ttccccgagg aagaggaggg cggctgtgag ctgagagtta agttcagcag gagcgccgac 1200

gcccctgcct accagcaagg agaatcaa ctgtacaacg agctgaacct gggcagacgg 1260

gaggaatacg atgtgctgga caagaggaga ggcagagacc ccgagatggg cggcaaacct 1320

agaagaaaga accccagga 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

Polynucleotides

<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 gttatactgccg 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 gaggaccaa 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 aggggcctgta taacgagctc 1320

cagaaggaca agatggccga ggcctacagc gagatcggca tgaagggcga aagaagaaga 1380

ggcaagggcc acgacggcct ctaccagggc ttaagcacag ctacaaagga cacctacgac 1440

gccctgcaca tgcaggcct gccccctaga tgattaatta aatcgat 1487

<210> 289

<211> 1502

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

Polynucleotides

<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 gagacccga gatgggcggc aaacctagaa gaaagaaccc ccaggagggc 1320

ctgtataacg agctccagaa ggacaagatg gccgaggcct acagcgagat cggcatgaag 1380

ggcgaaagaa gaagaggcaa gggccacgac ggcctctacc agggcttaag cacagctaca 1440

aaggacacct acgacgcct gcacatgcag gccctgcccc ctagatgatt aattaaatcg 1500

at 1502

<210> 290

<211> 1508

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

Polynucleotides

<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 gttatactgccg 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 tctaccagggg 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

Polynucleotides

<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 ggagaattggg 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 acacagcc 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 aggggagca 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 gacggggagga atacgatgtg 2820

ctggacaaga ggagaggcag agaccccgag atgggcggca aacctagaag aaagaacccc 2880

caggagggcc tgtataacga gctccagaag gacaagatgg ccgaggccta cagcgagatc 2940

ggcatgaagg gcgaaagaag aagaggcaag ggccacgacg gcctctacca gggcttaagc 3000

acagctacaa aggacacccta cgacgccctg cacatgcagg ccctgccccc tagatga 3057

<210> 292

<211> 1018

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthesis

polypeptide

<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 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 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 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 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 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 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 Cys Ala Arg Glu

610 615 620

Thr Asp Tyr Ser Ser Gly Met Gly Tyr 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 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 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

Polynucleotides

<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 ttggggagtat 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 gccgtttat 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 gccccacccac 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 agatggggat 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

polypeptide

<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 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 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

polypeptide

<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

polypeptide

<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

polypeptide

<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

polypeptide

<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 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 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

polypeptide

<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

polypeptide

<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

polypeptide

<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 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

polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(50)

<223> This sequence can encompass 1-10 "Pro Ala Pro Ala Pro" sequences.

Repeating unit

<400> 312

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

1 5 10 15

Ala Pro Ala Pro Pro Pro Ala Pro Ala Pro 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

polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(50)

<223> This sequence can encompass 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 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 (26)

1. An antibody or antigen-binding fragment thereof containing an anti-CD20 binding motif, said antibody or antigen-binding fragment comprising a heavy chain variable domain containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 and a light chain variable domain containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein According to the Kabat number, HCDR1 is shown as the amino acid sequence of SEQ ID NO:70; HCDR2 is shown as the amino acid sequence of SEQ ID NO:73; HCDR3 is shown as the amino acid sequence of SEQ ID NO:76; LCDR1 is shown as the amino acid sequence of SEQ ID NO:81; LCDR2 is shown as the amino acid sequence of SEQ ID NO:84; and LCDR3 is shown as the amino acid sequence of SEQ ID NO:87. 2. The antibody of claim 1 or its antigen-binding fragment, wherein: The heavy chain variable domain is shown in the amino acid sequence of SEQ ID NO:67, and the light chain variable domain is shown in the amino acid sequence of SEQ ID NO:78. 3. The antibody of claim 1 or 2 or its antigen-binding fragment, wherein the heavy chain complementarity-determining region and the light chain complementarity-determining region are within a single polypeptide. 4. The antibody of claim 1 or 2 or an antigen-binding fragment thereof, wherein the heavy chain complementarity-determining region is comprised of a first polypeptide and the light chain complementarity-determining region is comprised of a second polypeptide. 5. The antibody of claim 4 or an antigen-binding fragment thereof, wherein the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain. 6. The antibody of claim 1 or 2 or its antigen-binding fragment, further comprising an anti-CD19 binding motif. 7. The antibody of claim 6 or its antigen-binding fragment, wherein the anti-CD19 binding motif comprises a heavy chain variable domain including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 and a light chain variable domain including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: The HCDR1 is shown in SEQ ID NO:224, the HCDR2 is shown in SEQ ID NO:227, the HCDR3 is shown in SEQ ID NO:230, the LCDR1 is shown in SEQ ID NO:235, the LCDR2 is shown in SEQ ID NO:238, and the LCDR3 is shown in SEQ ID NO:241. 8. The antibody of claim 7 or its antigen-binding fragment, wherein the heavy chain variable domain of the anti-CD19 binding motif is as shown in the amino acid sequence of SEQ ID NO:221 and the light chain variable domain of the anti-CD19 binding motif is as shown in the amino acid sequence of SEQ ID NO:232. 9. The antibody of claim 7 or its antigen-binding fragment, wherein the heavy chain complementarity-determining region and the light chain complementarity-determining region of the anti-CD19 binding motif are within a single polypeptide. 10. The antibody of claim 7 or its antigen-binding fragment, wherein the heavy chain complementarity-determining region of the anti-CD20 binding motif, the light chain complementarity-determining region of the anti-CD20 binding motif, the heavy chain complementarity-determining region of the anti-CD19 binding motif, and the light chain complementarity-determining region of the anti-CD19 binding motif are within a single polypeptide. 11. The antibody of claim 1 or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment is contained in a chimeric antigen receptor. 12. The antibody of claim 11 or an antigen-binding fragment thereof, wherein the chimeric antigen receptor comprises a transmembrane domain, which is a transmembrane domain of 4-1BB, 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, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the zeta chain of a T-cell receptor, or any combination thereof. 13. The antibody of claim 7 or the antigen-binding fragment thereof, wherein (i) the heavy chain complementarity-determining region and the light chain complementarity-determining region of the anti-CD20 binding motif are located in a first polypeptide, and (ii) the heavy chain complementarity-determining region and the light chain complementarity-determining region of the anti-CD19 binding motif are located in a second polypeptide. 14. The antibody of claim 13 or an antigen-binding fragment thereof, wherein the first polypeptide is contained in a first chimeric antigen receptor. 15. The antibody of claim 13 or an antigen-binding fragment thereof, wherein the second polypeptide is contained in a second chimeric antigen receptor. 16. A nucleic acid encoding an antibody or an antigen-binding fragment thereof of any one of claims 1-15. 17. A vector comprising the nucleic acid of claim 16. 18. A method for generating engineered cells, the method comprising transfecting or transducing cells with a nucleic acid according to claim 16 or a vector according to claim 17. 19. A cell encoding or expressing an antibody or an antigen-binding fragment thereof of any one of claims 1-15. 20. The cell of claim 19, wherein the cell is an immune cell. 21. The cell of claim 19, wherein the cell is a T cell. 22. Use of a cell therapy composition comprising one or more cells in the preparation of a medicament for treating cancer in a subject in need, wherein the cells encode or comprise an antibody or an antigen-binding fragment thereof of any one of claims 1-15, wherein the cancer is leukemia, lymphoma, or myeloma. 23. Use of a cell therapy composition comprising one or more cells in the preparation of a medicament for immunizing a subject against cancer, wherein the cells encode or comprise an antibody or an antigen-binding fragment thereof of any one of claims 1-15, wherein the cancer is leukemia, lymphoma, or myeloma. 24. The use of claim 22 or 23, wherein the cell is a CAR-T cell. 25. The use of claim 22 or 23, wherein the cancer is chronic or acute leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, asymptomatic myeloma, multiple myeloma, or solitary myeloma. 26. The use of claim 22 or 23, wherein the cell therapy is an allogeneic cell therapy or an autologous cell therapy.