Chimeric antigens and T cell receptors and methods of use

Abstract

A chimeric antigen receptor (CAR) or T cell receptor (TCR) comprising one or more antigen binding motifs disclosed herein is provided. Aspects of the present disclosure relate to polynucleotides encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) comprising one or more antigen binding motifs. Antibodies and antigen binding systems comprising binding motifs that bind to CD20 and optionally binding motifs that bind to CD19 are provided, as well as methods of producing and using the same. The antibodies and antigen binding systems of the present disclosure comprise a CAR comprising an anti-CD20 binding motif and an anti-CD19 binding motif. Compositions, such as antibodies and CARs, as or comprising the anti-CD20 / anti-CD19 antigen binding systems of the present disclosure, are provided, as well as cell therapies comprising the same, which are useful, for example, in the treatment of cancer.

Description

[0001] This application is a divisional application based on a patent application with an application date of December 11, 2019, a priority date of December 12, 2018, an application number of 201980091506.9, and an invention name of “Chimeric Antigens and T Cell Receptors and Methods of Use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 778,893, filed on December 12, 2018, which is hereby incorporated by reference in its entirety. Background of the Invention

[0005] Human cancers, by their very nature, consist of normal cells that have undergone genetic or epigenetic transformation to become 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 cancer cells.

[0006] Current therapy T cell therapy relies on enriched or modified human T cells to target and kill cancer cells in patients. In order to increase the ability of T cells to target and kill specific cancer cells, methods have been developed to engineer T cells to express constructs that direct T cells to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which include binding domains that can interact with specific tumor antigens, allow T cells to target and kill cancer cells expressing specific tumor antigens. There is a demand for CARs and TCRs for targeting and killing cancer cells. SUMMARY OF THE INVENTION

[0008] In at least a first aspect, the present disclosure includes an antigen binding system, an antibody or an antigen binding fragment thereof comprising an anti-CD20 binding motif, wherein the anti-CD20 binding motif comprises the sequence of three heavy chain complementarity determining regions (HCDRs) of any one heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 1, 23, 45, 67, 89, 111, 133, 155, 177 and 199 and the sequence of three light chain CDRs (LCDRs) of any one light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210. In some embodiments, the anti-CD20 binding motif comprises a first domain comprising three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) and a second domain comprising three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3), wherein (i) HCDR1 has a sequence according to any one of SEQ ID NOs: 3-5, 25-27, 47-49, 69-71, 91-93, 113-115, 135-137, 157-159, 179-181; and 201-203; (ii) HCDR2 has a sequence according to any one of SEQ ID NOs: 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184, and 204-206; and (iii) HCDR3 has a sequence according to any one of SEQ ID NOs: NO:9-11, 31-33, 53-55, 75-77, 97-99, 119-121, 141-143, 163-165, 185-187 and 207-209; (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: NO:17-19, 39-41, 61-63, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and (vi) LCDR3 has a sequence according to 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 embodiments, the HCDR comprises: (i) a HCDR1 according to any one of SEQ ID NO:3-5;HCDR2 according to any one of SEQ ID NOs: 6-8; HCDR3 according to any one of SEQ ID NOs: 9-11; (ii) HCDR1 according to any one of SEQ ID NOs: 25-27; HCDR2 according to any one of SEQ ID NOs: 28-30; HCDR3 according to any one of SEQ ID NOs: 31-33; (iii) HCDR1 according to any one of SEQ ID NOs: 47-49; HCDR2 according to any one of SEQ ID NOs: 50-52; HCDR3 according to any one of SEQ ID NOs: 53-55; (iv) HCDR1 according to any one of SEQ ID NOs: 69-71; HCDR2 according to any one of SEQ ID NOs: 72-74; HCDR3 according to any one of SEQ ID NOs: 75-77; (v) HCDR1 according to any one of SEQ ID NOs: 91-93; HCDR2 according to any one of SEQ ID NOs: 94-96; HCDR3 according to any one of SEQ ID NOs: 141-143; (viii) HCDR1 according to any one of SEQ ID NOs: 157-159; HCDR2 according to any one of SEQ ID NOs: 160-162; HCDR3 according to any one of SEQ ID NOs: 163-165; (ix) HCDR1 according to any one of SEQ ID NOs: 179-181; HCDR2 according to any one of SEQ ID NOs: 180-183; HCDR3 according to any one of SEQ ID NOs: 184-185; (ix) HCDR1 according to any one of SEQ ID NOs: 190-201; HCDR2 according to any one of SEQ ID NOs: 202-203; HCDR3 according to any one of SEQ ID NOs: 204-205; (ix) HCDR1 according to any one of SEQ ID NOs: 191-202; HCDR2 according to any one of SEQ ID NOs: 205-206; HCDR3 according to any one of SEQ ID NOs: 207-208; NO: 182-184; HCDR2 according to any one of SEQ ID NO: 185-187; or (x) HCDR1 according to any one of SEQ ID NO: 201-203; HCDR2 according to any one of SEQ ID NO: 204-206; HCDR3 according to any one of SEQ ID NO: 207-209; and the LCDR comprises: (i) LCDR1 according to any one of SEQ ID 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 NOs:36-38; LCDR2 according to any one of SEQ ID NOs:39-41; LCDR3 according to any one of SEQ ID NOs:42-44; (iii) LCDR1 according to any one of SEQ ID NOs:58-60; LCDR2 according to any one of SEQ ID NOs:61-63; LCDR3 according to any one of SEQ ID NOs:64-66; (iv) LCDR1 according to any one of SEQ ID NOs:80-82; LCDR2 according to any one of SEQ ID NOs:83-85; LCDR3 according to any one of SEQ ID NOs:86-88; (v) LCDR1 according to any one of SEQ ID NOs:102-104; LCDR2 according to any one of SEQ ID NOs:105-107; LCDR3 according to any one of SEQ ID NOs:108-110; (vi) LCDR1 according to any one of SEQ ID NOs:111-112; a LCDR1 according to any one of SEQ ID NOs: 124-126; a LCDR2 according to any one of SEQ ID NOs: 127-129; a LCDR3 according to any one of SEQ ID NOs: 130-132; (vii) a LCDR1 according to any one of SEQ ID NOs: 146-148; a LCDR2 according to any one of SEQ ID NOs: 149-151; a LCDR3 according to any one of SEQ ID NOs: 152-154; (viii) a LCDR1 according to any one of SEQ ID NOs: 168-170; a LCDR2 according to any one of SEQ ID NOs: 171-173; a LCDR3 according to any one of SEQ ID NOs: 174-176; (ix) a LCDR1 according to any one of SEQ ID NOs: 190-192; a LCDR2 according to any one of SEQ ID NOs: 193-195; a LCDR3 according to any one of SEQ ID NOs: or (x) a LCDR1 according to any one of SEQ ID NOs: 212-214; a LCDR2 according to any one of SEQ ID NOs: 215-217; or a LCDR3 according to any one of SEQ ID NOs: 218-220.

[0009] In various embodiments, the antigen binding system, antibody or antigen binding fragment thereof of the present disclosure comprises a first domain comprising three heavy chain complementary determining regions (HCDRs) and a second domain comprising three light chain complementary determining regions (LCDRs), wherein: the HCDRs and LCDRs comprise: (i) a HCDR1 according to any one of SEQ ID NOs: 3-5; a HCDR2 according to any one of SEQ ID NOs: 6-8; a HCDR3 according to any one of SEQ ID NOs: 9-11; a LCDR1 according to any one of SEQ ID NOs: 14-16; a LCDR2 according to any one of SEQ ID NOs: 17-19; a LCDR3 according to any one of SEQ ID NOs: 20-22; (ii) a HCDR1 according to any one of SEQ ID NOs: 25-27; a HCDR2 according to any one of SEQ ID NOs: 28-30; a HCDR3 according to any one of SEQ ID NOs: 31-33; a LCDR1 according to any one of SEQ ID NOs: 36-38; a LCDR2 according to any one of SEQ ID NOs: 37-39; a LCDR3 according to any one of SEQ ID NOs: 40-41; a LCDR3 according to any one of SEQ ID NOs: 42-43; a LCDR3 according to any one of SEQ ID NOs: 44-45; a LCDR3 according to any one of SEQ ID NOs: 45-46; a LCDR3 according to any one of SEQ ID NOs: 47-48; a LCDR3 according to any one of SEQ ID NOs: 49-50; a LCDR1 according to any one of SEQ ID NOs: 51-52; a LCDR1 according to any one of SEQ ID NOs: 52-54; a LCDR1 according to any one of SEQ ID NOs: 53-55; a LCDR2 according to any one of SEQ ID NOs:39-41; a LCDR3 according to any one of SEQ ID NOs:42-44; (iii) a HCDR1 according to any one of SEQ ID NOs:47-49; a HCDR2 according to any one of SEQ ID NOs:50-52; a HCDR3 according to any one of SEQ ID NOs:53-55; a LCDR1 according to any one of SEQ ID NOs:58-60; a LCDR2 according to any one of SEQ ID NOs:61-63; a LCDR3 according to any one of SEQ ID NOs:64-66; (iv) a HCDR1 according to any one of SEQ ID NOs:69-71; a HCDR2 according to any one of SEQ ID NOs:72-74; a HCDR3 according to any one of SEQ ID NOs:75-77; a LCDR1 according to any one of SEQ ID NOs:80-82; a LCDR2 according to any one of SEQ ID NOs:83-85; a LCDR3 according to any one of SEQ ID NOs:86-87; 104-107; LCDR3 according to any one of SEQ ID NOs: 108-110; (vi) HCDR1 according to any one of SEQ ID NOs: 113-115;146-148; LCDR2 according to any one of SEQ ID NOs: 149-151; LCDR3 according to any one of SEQ ID NOs: 152-154; (viii) HCDR1 according to any one of SEQ ID NOs: 147-149; LCDR2 according to any one of SEQ ID NOs: 151-152; LCDR3 according to any one of SEQ ID NOs: 153-154; (viii) HCDR1 according to any one of SEQ ID NOs: 148-149; LCDR2 according to any one of SEQ ID NOs: 152-154; (viii) HCDR2 according to any one of SEQ ID NOs: 149-151; LCDR3 according to any one of SEQ ID NOs: 153-154; (viii) HCDR1 according to any one of SEQ ID NOs: 147-149; LCDR2 according to any one of SEQ ID NOs: 154-155; (viii) HCDR2 according to any one of SEQ ID NOs: 148-149; LCDR3 according to any one of SEQ ID NOs: 155-156; (viii) HCDR2 according to any one of SEQ ID NOs: 149-151; LCDR3 according to any one of SEQ ID NOs: 156-155; (viii) HCDR2 according to any one of SEQ ID NOs: 147-149; LCDR2 according to any one of SEQ ID NOs: 157-158; (viii) HCDR2 according to any one of SEQ ID NOs: 148-149; LCDR3 according to any one of SEQ ID NOs: 159-160; (viv) HCDR2 according to any one of SEQ ID NOs: 149-1 a LCDR1 according to any one of SEQ ID NOs: 190-192; a LCDR2 according to any one of SEQ ID NOs: 193-195; a LCDR3 according to any one of SEQ ID NOs: 196-198; or (x) a HCDR1 according to any one of SEQ ID NOs: 191-192; a LCDR2 according to any one of SEQ ID NOs: 193-195; a LCDR3 according to any one of SEQ ID NOs: 196-198; or (x) a HCDR1 according to any one of SEQ ID NOs: 192-193; a LCDR2 according to any one of SEQ ID NOs: 195-196; a LCDR3 according to any one of SEQ ID NOs: 197-198; or (x) a HCDR1 according to any one of SEQ ID NOs: 193-195; a LCDR2 according to any one of SEQ ID NOs: 196-198; or HCDR1 according to any one of SEQ ID NOs: 201-203; HCDR2 according to any one of SEQ ID NOs: 204-206; HCDR3 according to any one of SEQ ID NOs: 207-209; LCDR1 according to any one of SEQ ID NOs: 212-214; LCDR2 according to any one of SEQ ID NOs: 215-217;LCDR3 according to any one of SEQ ID NOs: 218-220. In some embodiments, the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1, 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: 177 or SEQ ID NO: 199; and (ii) the light chain variable 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 comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1 and the light chain variable domain is at least 80% identical to SEQ ID NO: 12; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 23 and the light chain variable domain is at least 80% identical to SEQ ID NO: 34; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 45 and the light chain variable domain is at least 80% identical to SEQ ID NO: 56; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 67 and the light chain variable domain is at least 80% identical to SEQ ID NO: 78; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 89 and the light chain variable domain is at least 80% identical to SEQ ID NO: 100; (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 101 and the light chain variable domain is at least 80% identical to SEQ ID NO: 102; NO:111 is at least 80% identical and the light chain variable domain is at least 80% identical to SEQ ID NO:122; (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:133 and the light chain variable domain is at least 80% identical to SEQ ID NO:144; (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:155 and the light chain variable domain is at least 80% identical to SEQ ID NO:166;(ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 177 and the light chain variable domain is at least 80% identical to SEQ ID NO: 188; or (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 199 and the light chain variable domain is at least 80% identical to SEQ ID NO: 210.;

[0010] In some embodiments of the present disclosure comprising three HCDRs and three LCDRs, the three HCDRs and the three LCDRs are comprised by a single polypeptide. In some embodiments of the present disclosure comprising three HCDRs and three LCDRs, the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide. In some embodiments, the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain.

[0011] In some embodiments, the antigen binding system, antibody or antigen binding fragment thereof further comprises: (i) a binding motif that binds to an antigen selected from the group consisting of 5T4, alpha-fetoprotein, B cell maturation antigen (BCMA), B cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, 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 gpl20, IL-1Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B cell activating factor receptor) and combinations thereof; and / or (ii) binding motifs for 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 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 NOs: 223-225; HCDR2 according to any one of SEQ ID NOs: 226-228; HCDR3 according to any one of SEQ ID NOs: 229-231; LCDR1 according to any one of SEQ ID NOs: 234-236; LCDR2 according to any one of SEQ ID NOs: 237-239; LCDR3 according to any one of SEQ ID NOs: 240-242. In some embodiments, the anti-CD19 binding motif comprises a first heavy chain variable domain comprising three HCDRs of the anti-CD19 binding motif and a light chain variable domain comprising 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 comprised by 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 comprised by a single polypeptide.

[0012] In various embodiments, the antigen binding system, antibody, or antigen binding fragment thereof is a chimeric antigen receptor or is comprised by a chimeric antigen receptor. In some embodiments, the antigen binding system, antibody, or antigen binding fragment thereof is a single polypeptide, which is a chimeric antigen receptor or is comprised by a chimeric antigen receptor, and the chimeric antigen receptor is a bispecific chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain, i.e., 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 transmembrane domain of a zeta chain of a 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 a 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 comprised by a different second polypeptide. In some embodiments, the first polypeptide is a first chimeric antigen receptor or is comprised by a first chimeric antigen receptor. In some embodiments, the second polypeptide is a second chimeric antigen receptor or is comprised by a second chimeric antigen receptor.

[0013] In various embodiments, the disclosure comprises nucleic acids encoding at least one polypeptide of the disclosure and / or vectors comprising such nucleic acids. The disclosure further comprises methods for generating engineered cells, the methods comprising transfecting or transducing cells with nucleic acids encoding at least one polypeptide of the disclosure. Further provided herein are cells encoding or expressing the antigen binding systems, antibodies or antigen binding fragments provided herein, optionally wherein the cells are immune cells, optionally wherein the cells are T cells.

[0014] The present disclosure further includes a method for 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 the present disclosure. Also provided herein is a method for inducing an immune response in a subject or immunizing a subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising an antigen binding system, antibody, or antigen binding fragment thereof of the present disclosure. In some embodiments, the cell is a CAR-T cell. In various embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic 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 conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma) ), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma or Waldenstrom's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, hairy cell lymphoma / leukemia, or a combination thereof. In some embodiments, the cell therapy is an allogeneic cell therapy or an autologous cell therapy.

[0015] At least one aspect of the present disclosure comprises a chimeric antigen receptor comprising an anti-CD20 binding motif comprising three heavy chain complementarity determining regions (HCDRs) and three light chain CDRs (LCDRs), wherein the three HCDRs are contained within a heavy chain variable region (HCVR) sequence selected from the group consisting of SEQ ID NOs: 1, 23, 45, 67, 89, 111, 133, 155, 177 and 199, and the three LCDRs are 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 complementary determining regions (HCDRs) and the second domain comprises three light chain complementary determining regions (LCDRs), wherein (i) HCDR1 has a sequence according to any one of SEQ ID NOs: 3-5, 25-27, 47-49, 69-71, 91-93, 113-115, 135-137, 157-159, 179-181; and 201-203; (ii) HCDR2 has a sequence according to any one of SEQ ID NOs: 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184; and 204-206; (iii) 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, 185-187; and 207-209; (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, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and (vi) LCDR3 has a sequence according to 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 embodiments, the HCDR comprises: (i) a HCDR1 according to any one of SEQ ID NO:3-5; a HCDR2 according to any one of SEQ ID NO:6-8; a HCDR3 according to any one of SEQ ID NO:9-11;(ii) a HCDR1 according to any one of SEQ ID NOs: 25-27; a HCDR2 according to any one of SEQ ID NOs: 28-30; a HCDR3 according to any one of SEQ ID NOs: 31-33; (iii) a HCDR1 according to any one of SEQ ID NOs: 47-49; a HCDR2 according to any one of SEQ ID NOs: 50-52; a HCDR3 according to any one of SEQ ID NOs: 53-55; (iv) a HCDR1 according to any one of SEQ ID NOs: 69-71; a HCDR2 according to any one of SEQ ID NOs: 72-74; a HCDR3 according to any one of SEQ ID NOs: 75-77; (v) a HCDR1 according to any one of SEQ ID NOs: 91-93; a HCDR2 according to any one of SEQ ID NOs: 94-96; a HCDR3 according to any one of SEQ ID NOs: 97-99; (vi) a HCDR1 according to any one of SEQ ID NOs: 91-93; a HCDR2 according to any one of SEQ ID NOs: 94-96; a HCDR3 according to any one of SEQ ID NOs: 97-99; 157-159; HCDR2 according to any one of SEQ ID NOs: 160-162; HCDR3 according to any one of SEQ ID NOs: 163-165; (ix) HCDR1 according to any one of SEQ ID NOs: 179-181; HCDR2 according to any one of SEQ ID NOs: 182-184; HCDR3 according to any one of SEQ ID NOs: 185-186; (ix) HCDR1 according to any one of SEQ ID NOs: 183-187; HCDR2 according to any one of SEQ ID NOs: 187-188; HCDR3 according to any one of SEQ ID NOs: 189-200; (ix) HCDR1 according to any one of SEQ ID NOs: 190-201; HCDR2 according to any one of SEQ ID NOs: 191-202; HCDR3 according to any one of SEQ ID NOs: 192-203; (ix) HCDR1 according to any one of SEQ ID NOs: 193-204; HCDR2 according to any one of SEQ ID NOs: 194-205; NO:185-187; or (x) HCDR1 according to any one of SEQ ID NO:201-203; HCDR2 according to any one of SEQ ID NO:204-206; HCDR3 according to any one of SEQ ID NO:207-209; and the LCDR comprises: (i) LCDR1 according to any one of SEQ ID 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;a LCDR3 according to any one of SEQ ID NOs: 42-44; (iii) a LCDR1 according to any one of SEQ ID NOs: 58-60; a LCDR2 according to any one of SEQ ID NOs: 61-63; a LCDR3 according to any one of SEQ ID NOs: 64-66; (iv) a LCDR1 according to any one of SEQ ID NOs: 80-82; a LCDR2 according to any one of SEQ ID NOs: 83-85; a LCDR3 according to any one of SEQ ID NOs: 86-88; (v) a LCDR1 according to any one of SEQ ID NOs: 102-104; a LCDR2 according to any one of SEQ ID NOs: 105-107; a LCDR3 according to any one of SEQ ID NOs: 108-110; (vi) a LCDR1 according to any one of SEQ ID NOs: 124-126; a LCDR2 according to any one of SEQ ID NOs: 127-129; a LCDR3 according to any one of SEQ ID NOs: a LCDR1 according to any one of SEQ ID NOs: 190-192; a LCDR2 according to any one of SEQ ID NOs: 193-195; a LCDR3 according to any one of SEQ ID NOs: 196-198; or (x) a LCDR1 according to any one of SEQ ID NOs: 212-214; a LCDR2 according to any one of SEQ ID NOs: 215-216; a LCDR3 according to any one of SEQ ID NOs: 217-218; a LCDR2 according to any one of SEQ ID NOs: 219-220; a LCDR3 according to any one of SEQ ID NOs: 221-224; a LCDR3 according to any one of SEQ ID NOs: 222-226; a LCDR3 according to any one of SEQ ID NOs: 223-227; a LCDR3 according to any one of SEQ ID NOs: 224-228; a LCDR3 according to any one of SEQ ID NOs: 225-229; a LCDR1 according to any one of SEQ ID NOs: 226-229; a LCDR2 according to any one of SEQ ID NOs: 227-228; a LCDR3 according to any one of SEQ ID NOs: 228-229; LCDR2 according to any one of SEQ ID NOs: 215-217; LCDR3 according to any one of SEQ ID NOs: 218-220.;

[0016] In some embodiments, the chimeric antigen receptor comprises a first domain comprising three heavy chain complementary determining regions (HCDRs) and a second domain comprising three light chain complementary 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) a HCDR1 according to any one of SEQ ID NOs: 3-5; a HCDR2 according to any one of SEQ ID NOs: 6-8; a HCDR3 according to any one of SEQ ID NOs: 9-11; a LCDR1 according to any one of SEQ ID NOs: 14-16; a LCDR2 according to any one of SEQ ID NOs: 17-19; a LCDR3 according to any one of SEQ ID NOs: 20-22; (ii) a HCDR1 according to any one of SEQ ID NOs: 25-27; a HCDR2 according to any one of SEQ ID NOs: 28-30; a LCDR3 according to any one of SEQ ID NOs: 31-33; LCDR3 according to any one of SEQ ID NOs:36-38; LCDR1 according to any one of SEQ ID NOs:39-41; LCDR3 according to any one of SEQ ID NOs:42-44; (iii) HCDR1 according to any one of SEQ ID NOs:47-49; HCDR2 according to any one of SEQ ID NOs:50-52; HCDR3 according to any one of SEQ ID NOs:53-55; LCDR1 according to any one of SEQ ID NOs:58-60; LCDR2 according to any one of SEQ ID NOs:61-63; LCDR3 according to any one of SEQ ID NOs:64-66; (iv) HCDR1 according to any one of SEQ ID NOs:69-71; HCDR2 according to any one of SEQ ID NOs:72-74; HCDR3 according to any one of SEQ ID NOs:75-77; LCDR3 according to any one of SEQ ID NOs:78-79; a LCDR1 according to any one of SEQ ID NOs: 80-82; a LCDR2 according to any one of SEQ ID NOs: 83-85; a LCDR3 according to any one of SEQ ID NOs: 86-88; (v) a HCDR1 according to any one of SEQ ID NOs: 91-93; a HCDR2 according to any one of SEQ ID NOs: 94-96; a HCDR3 according to any one of SEQ ID NOs: 97-99; a LCDR1 according to any one of SEQ ID NOs: 102-104; a LCDR2 according to any one of SEQ ID NOs: 105-107; a LCDR3 according to any one of SEQ ID NOs: 108-110;(vi) a HCDR1 according to any one of SEQ ID NOs: 113-115; a HCDR2 according to any one of SEQ ID NOs: 116-118; a HCDR3 according to any one of SEQ ID NOs: 119-121; a LCDR1 according to any one of SEQ ID NOs: 124-126; a LCDR2 according to any one of SEQ ID NOs: 127-129; a LCDR3 according to any one of SEQ ID NOs: 130-132; (vii) a HCDR1 according to any one of SEQ ID NOs: 135-137; a HCDR2 according to any one of SEQ ID NOs: 138-140; a HCDR3 according to any one of SEQ ID NOs: 141-143; a LCDR1 according to any one of SEQ ID NOs: 146-148; a LCDR2 according to any one of SEQ ID NOs: 149-151; a LCDR2 according to any one of SEQ ID NOs: 152-153; a LCDR3 according to any one of SEQ ID NOs: 154-155; a LCDR3 according to any one of SEQ ID NOs: 156-157; a LCDR2 according to any one of SEQ ID NOs: 157-158; a LCDR3 according to any one of SEQ ID NOs: 159-160; a LCDR3 according to any one of SEQ ID NOs: 161-162; a LCDR1 according to any one of SEQ ID NOs: 163-164; a LCDR1 according to any one of SEQ ID NOs: 165-166 a LCDR1 according to any one of SEQ ID NOs: 190-192; a LCDR2 according to any one of SEQ ID NOs: 193-195; a LCDR3 according to any one of SEQ ID NOs: 194-196; (ix) a HCDR1 according to any one of SEQ ID NOs: 190-192; a LCDR2 according to any one of SEQ ID NOs: 193-195; a LCDR3 according to any one of SEQ ID NOs: 196-197; a LCDR1 according to any one of SEQ ID NOs: 197-198; a LCDR2 according to any one of SEQ ID NOs: 199-200; a LCDR3 according to any one of SEQ ID NOs: 201-203; a LCDR3 according to any one of SEQ ID NOs: 204-205; a LCDR1 according to any one of SEQ ID NOs: 205-206; a LCDR1 according to any one of SEQ ID NOs: 207-208; a LCDR2 according to any one of SEQ ID NOs: 209-210; a LCDR3 according to any one of SEQ ID NOs: 201-211; a LCDR3 according to any one of SEQ ID NOs: 203-213; a LCDR3 according to any one of SEQ ID NOs: 204-214; a LCDR3 according to any one of SEQ ID NOs: 196-198; or (x) a HCDR1 according to any one of SEQ ID NOs: 201-203; a HCDR2 according to any one of SEQ ID NOs: 204-206; a HCDR3 according to any one of SEQ ID NOs: 207-209; a LCDR1 according to any one of SEQ ID NOs: 212-214; a LCDR2 according to any one of SEQ ID NOs: 215-217;LCDR3 according to any one of SEQ ID NOs: 218 to 220. In various embodiments, the chimeric antigen receptor comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1, 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: 177 or SEQ ID NO: 199; and (ii) the light chain variable 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 comprises a first heavy chain variable domain comprising three HCDRs and a light chain variable domain comprising three LCDRs, wherein: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1, and the light chain variable domain is at least 80% identical to SEQ ID NO: 12; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 23, and the light chain variable domain is at least 80% identical to SEQ ID NO: 34; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 45, and the light chain variable domain is at least 80% identical to SEQ ID NO: 56; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 67, and the light chain variable domain is at least 80% identical to SEQ ID NO: 78; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 89, and the light chain variable domain is at least 80% identical to SEQ ID NO: 100; (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: ID NO:111 is at least 80% identical, and the light chain variable domain is at least 80% identical to SEQ ID NO:122; (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:133, and the light chain variable domain is at least 80% identical to SEQ ID NO:144; (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:155, and the light chain variable domain is at least 80% identical to SEQ ID NO:166;(ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 177, and the light chain variable domain is at least 80% identical to SEQ ID NO: 188; or (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 199, and the light chain variable domain is at least 80% identical to SEQ ID NO: 210.;

[0017] In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs and the three LCDRs are comprised by a single polypeptide. In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide. In some embodiments, the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain. In some embodiments, the chimeric antigen receptor further comprises: (i) a binding motif that specifically binds to an antigen selected from the group consisting of 5T4, alpha-fetoprotein, B cell maturation antigen (BCMA), B cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HER1-HER2 group In some embodiments, the chimeric antigen receptor further comprises an anti-CD19 binding motif.

[0018] 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 NOs: 223-225; HCDR2 according to any one of SEQ ID NOs: 226-228; HCDR3 according to any one of SEQ ID NOs: 229-231; LCDR1 according to any one of SEQ ID NOs: 234-236; LCDR2 according to any one of SEQ ID NOs: 237-239; LCDR3 according to any one of SEQ ID NOs: 240-242. In some embodiments, the anti-CD19 binding motif comprises a first heavy chain variable domain comprising the three HCDRs of the anti-CD19 binding motif and a light chain variable domain comprising the 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 comprised by a single polypeptide.

[0019] 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 LCDR motifs of the anti-CD19 binding motif are together comprised by a single polypeptide. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that is a transmembrane domain of 4-1BB / CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.

[0020] In various embodiments, the present disclosure comprises a bicistronic chimeric antigen receptor comprising a first chimeric antigen receptor of the present disclosure and a second chimeric antigen receptor comprising a binding motif that specifically binds to an antigen selected from the group consisting of 5T4, alpha-fetoprotein, B cell maturation antigen (BCMA), B cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, 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 gpl20, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B cell activating factor receptor) and combinations thereof; and / or a binding motif that specifically binds to a B cell characteristic antigen, optionally wherein the B cell characteristic antigen is not CD19 or CD20. In some embodiments, the second chimeric antigen receptor comprises an anti-CD19 binding motif.

[0021] The present disclosure further provides nucleic acids encoding at least one polypeptide of the present disclosure and / or vectors comprising such nucleic acids. The present disclosure also includes methods for generating engineered cells, the methods comprising transfecting or transducing cells with nucleic acids of the present disclosure. In various embodiments, the present disclosure includes cells encoding or expressing chimeric antigen receptors provided herein, optionally wherein the cells are immune cells, optionally wherein the cells are T cells.

[0022] The present disclosure further provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising a chimeric antigen receptor of the present disclosure. Further provided herein is a method for inducing an immune response in a subject or immunizing a subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising a chimeric antigen receptor of the present disclosure. In various embodiments, the cell is a CAR-T cell. In various embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic 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 conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma) ), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma or Waldenstrom's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, hairy cell lymphoma / leukemia, or a combination thereof. In some embodiments, the cell therapy is an allogeneic cell therapy or an autologous cell therapy.

[0023] In at least one aspect, the disclosure comprises 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 NOs: 1, 23, 45, 67, 89, 111, 133, 155, 177, 199, 12, 34, 56, 78, 100, 122, 144, 166, 188, and 210. In some embodiments, the CD19 binding motif comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 by a single polypeptide. In some embodiments, the anti-CD20 binding motif and the CD19 binding motif are comprised by different polypeptides.

[0024] In at least one aspect, the 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, the 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. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present disclosure relates to new polypeptides comprising new antigen binding molecules and polynucleotides encoding them. Some aspects of the present disclosure relate to polynucleotides encoding at least one chimeric antigen receptor (CAR) comprising a heavy chain and a light chain (or its CDR) disclosed herein. The present disclosure also provides vectors (e.g., viral vectors) comprising such polynucleotides and compositions comprising such vectors. The present disclosure further provides polynucleotides encoding such CAR or TCR and compositions comprising such polynucleotides. The present disclosure further provides engineered cells (e.g., T cells) comprising such polynucleotides and / or transduced with such viral vectors and compositions comprising such engineered cells. The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising multiple engineered T cells. The present 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, the present disclosure provides a method for inducing immunity against a tumor, comprising administering to a subject an effective amount of cells comprising polynucleotides, vectors, or polypeptides of the present disclosure. Other aspects of the present disclosure relate to cells comprising CAR and their use in T cell therapy for treating patients with cancer.

[0027] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. Although the present disclosure has been described in conjunction with the detailed description of the present disclosure, the foregoing description is intended to illustrate rather than limit the scope of the present disclosure, and the scope of the present disclosure is limited 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 mentioned herein establish the 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 disclosed foreign patents and patent applications cited herein are incorporated herein by reference. All other disclosed references, dictionaries, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. From the following detailed description including embodiments and claims, other features and advantages of the present disclosure will be apparent.

[0028] definition

[0029] In order to more easily understand the present disclosure, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0030] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] As used herein, the term "or" should be construed as inclusive and encompasses both "or" and "and," unless expressly stated or obvious from the context.

[0032] As used herein, the term "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include A and B; A or B; A (alone); and B (alone). 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).

[0033] As used herein, the terms "eg" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly listed in the specification.

[0034] The terms “or more”, “at least”, “more than”, etc., 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 ,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129 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. Also included are any larger numbers or fractions therebetween.

[0035] Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0036] The terms “plurality”, “at least two”, “two or more”, “at least a second”, etc., shall be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 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 included are any larger numbers or fractions therebetween.

[0037] Throughout the specification, the word "comprising" or variations will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. It should be understood that wherever aspects are described herein with the language "comprising", other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0038] Unless expressly stated or apparent from the context, as used herein, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within or exceeding one standard deviation as practiced in the art. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ± 10%). Therefore, "about" can be understood as being greater than or less than the 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. In addition, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to 5 times. When a specific value or composition is provided in the present disclosure, unless otherwise stated, it should be assumed that "about" or "consisting essentially of" means within an acceptable error range for the specific value or composition.

[0039] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers).

[0040] The units, prefixes, and symbols used herein are provided in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the present disclosure relates. 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 many of the terms used in the present disclosure to those skilled in the art.

[0042] "Administering" refers to physically introducing a medicament into a subject using any of various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the preparation disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection and including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, via non-parenteral routes such as oral administration of the preparation. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or local. Administration can also be implemented, for example, once, many times, and / or in one or more long periods of time.

[0043] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into highly variable regions, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL comprises 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 antigens. The constant region of Ab can 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 tetrameric agents of about 150 kD, consisting of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each), which are associated with each other into a structure generally referred to as a "Y-shaped". The heavy and light chains are linked or connected to each other by a single disulfide bond; two additional disulfide bonds connect the heavy chain hinge regions to each other, thereby connecting the dimers to each other and forming a tetramer. Naturally produced antibodies are also glycosylated, for example, on the CH2 domain.

[0044] The term "human antibody" is intended to include antibodies having variable domain and constant domain sequences generated, assembled or derived from human immunoglobulin sequences, or sequences that are indistinguishable from them. In some embodiments, antibodies (or antibody components) may be considered "human" even if their amino acid sequences contain residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., variations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). The term "humanized" is intended to include antibodies having variable domains with sequences derived from variable domains of non-human species (e.g., mice) that have been modified to be more similar to sequences encoded by human germlines. In some embodiments, a "humanized" antibody comprises one or more framework domains having substantially the amino acid sequence of a human framework domain, and one or more complementary determining regions having substantially the same amino acid sequence as that of a non-human antibody. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant domain. In some embodiments, a humanized antibody may comprise the C of a human heavy chain constant domain.H 1. Hinge, C H 2. C H 3 and optionally C H 4.

[0045] The antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy 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, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab') fragments, 2 Fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to a polyclonal antibody population. The antibodies may also include, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single-chain Fvs, polypeptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof), camelid antibodies, single-chain or tandem diabodies Mini-antibodies, Ankyrin repeat protein or DART, TCR-like antibodies, MicroProteins and

[0046] Immunoglobulins can be derived from any known isotype, including but not limited to IgA, secretory IgA, IgG, IgE and IgM. IgG subclasses 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 the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, for example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal 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. In the absence of explicit instructions, unless otherwise indicated in the context, the term "antibody" also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes monovalent and divalent fragments, as well as single-chain Abs.

[0047] "Antigen binding molecule", "antigen binding portion" or "antibody fragment" refers to any molecule comprising an antigen binding portion (e.g., CDR) of an antibody from which the molecule is derived. Antigen binding molecules may include antigen complementary determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, antigen binding molecules bind to antigens on tumor cells. In some embodiments, antigen binding molecules bind to antigens on cells involved in hyperproliferative diseases or bind to viral or bacterial antigens. In certain embodiments, antigen binding molecules bind to BCMA, CLL-1 or FLT3. In certain embodiments, antigen binding molecules bind to CD19, CD20 or both. In further embodiments, antigen binding molecules are antibody fragments that specifically bind to antigens, including one or more complementary determining regions (CDRs). In further embodiments, antigen binding molecules are single-chain variable fragments (scFv). In some embodiments, the antigen binding molecule comprises or consists of an avimer.

[0048] In some cases, the sequence of the CDR and the CDR found in the reference antibody (e.g., antibody of the present disclosure) and / or the CDR provided in the present disclosure is substantially the same. In some embodiments, the CDR is substantially the same as the reference CDR (e.g., CDR provided in the present disclosure) because it is identical in sequence or contains 1, 2, 3, 4 or 5 (e.g., 1-5) amino acid substitutions compared to the reference CDR. In some embodiments, the CDR is substantially the same as the 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 (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) with the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR because it shows at least 96%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR because one amino acid within the CDR is deleted, added or substituted compared to the reference CDR, and the CDR has an amino acid sequence that is otherwise identical to the reference amino acid sequence. In some embodiments, the CDR is substantially identical to the reference CDR because 2, 3, 4 or 5 (e.g., 2-5) amino acids within the CDR are deleted, added or substituted compared to the reference CDR, and the CDR has an amino acid sequence that is otherwise identical to the reference CDR. In various embodiments, the antigen-binding fragment binds to the same antigen as the reference antibody.

[0049] Fabs can be produced by any means. For example, in some embodiments, Fabs can be produced enzymatically or chemically by fragmentation of complete antibodies. In some embodiments, Fabs can be produced recombinantly (i.e., by expression of engineered nucleic acid sequences). In some embodiments, Fabs can be produced by synthesis in whole or in part. In some embodiments, Fabs 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).

[0050] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, generally about 110 to 120 amino acids at the amino terminal in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which differ greatly in sequence between antibodies and are used for the binding and specificity of a specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions called complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are called framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies with antigens. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0051] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen binding molecule thereof.

[0052] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen binding molecule thereof.

[0053] Several definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on analysis of available complex crystal structures.

[0054] Table 1: CDR numbers

[0055]

[0056] The term "Kabat numbering" and similar terms are generally recognized in the art, and refer to the numbering system of amino acid residues in the heavy chain and light chain variable region of an antibody or its antigen binding molecules. In some aspects, 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, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDR in the heavy chain molecule of an antibody is usually present at amino acid positions 31 to 35 (which optionally can include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) after 35) (CDR1), amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In specific embodiments, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0057] In certain aspects, the CDRs of an antibody can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., 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. Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are 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 specific embodiments, the CDRs of the antibodies described herein have been identified according to the Chothia numbering scheme.

[0058] The terms "constant region" and "constant domain" are interchangeable and have common meanings in the art. The constant region is the portion of an antibody, such as the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in binding the antibody to an antigen, but may exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0059] When used in reference to antibodies, the term "heavy chain" can refer to any of the different types, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, such as IgG 1 IgG 2 IgG3 and IgG 4 .

[0060] When used in reference to antibodies, the term "light chain" can refer to any of the different types, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0061] "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 indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D 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 (K D ) and equilibrium association constant (K A ). D By k off / k on The quotient of K A By k on / k off Calculate the quotient of k on is, for example, the binding rate constant of an antibody to an antigen, while k off Refers to, for example, the dissociation of antibodies and antigens. on and k off The method can be performed by techniques known to those skilled in the art, such as or KinExA assay.

[0062] "Conservative amino acid substitution" refers to an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR or framework region of an antibody or its antigen binding molecule may be substituted with amino acid residues with similar side chains. In general, if two sequences contain conservative amino acid substitutions at corresponding positions, they are generally considered to be "substantially similar". For example, certain amino acids are often classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substituting one amino acid for another amino acid of the same type can be considered a conservative substitution. Exemplary amino acid classifications are summarized in Tables 2 and 3 below: Table 2

[0063]

[0064]

[0065] Table 3

[0066] Amino Acids 3 letters 1 Letter Asparagine or Aspartic Acid Asx B Glutamine or Glutamate Gx Z Leucine or Isoleucine Xle J Unassigned or unknown amino acid XA X

[0067] The term "heterologous" means from any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein (e.g., a corresponding human costimulatory protein) having an amino acid sequence of SEQ ID NO: 232 is an amino acid that does not naturally occur as a wild-type human costimulatory protein (i.e., does not align with it). For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than a wild-type human costimulatory protein coding sequence.

[0068] The term "epi-position" is a term in this area and refers to the local region of an antigen that an antibody can specifically bind. An epi-position can be, for example, the continuous amino acids (linear or continuous epi-position) of a polypeptide or an epi-position can be, for example, two or more discontinuous regions (conformation, non-linear, discontinuous or discontinuous epi-positions) from a polypeptide or a plurality of polypeptides. In certain embodiments, the epi-position of antibody binding can be determined by, for example, nuclear magnetic resonance spectroscopy (NMR spectroscopy), X-ray diffraction crystallography, ELISA, hydrogen / deuterium exchange and mass spectrometry (for example, liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (for example, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giege 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 known X-ray diffraction techniques, and can be refined using computer software such as X-PLOR (Yale University, 1992, marketed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; US 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 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 a description 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.

[0069] An antigen binding molecule, antibody or antigen binding molecule "cross-competes" with a reference antibody or antigen binding molecule if the interaction between the antigen and the first binding molecule, antibody or antigen binding molecule blocks, limits, inhibits or otherwise reduces the ability of the reference binding molecule, reference antibody or antigen binding molecule to interact with the antigen. Cross-competition can be complete, such as binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it can be partial, such as binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In certain embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds to the same or overlapping epitope as the reference antigen binding molecule. Many types of competitive binding assays can be used to determine whether one antigen binding molecule competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA); sandwich competition 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 label assay, solid phase direct label sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (Cheung, et al., 1986, J. Immunol. 137:3614-3619); al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0070] The term "binding" generally refers to a non-covalent association between two or more entities. Direct binding involves physical contact between the entities or moieties. "Indirect" binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts, for example, where the interacting entities or moieties are studied in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and / or in a biological system such as a cell).

[0071] As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex) as such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, immunoassays, KinEx A3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In a specific embodiment, the molecule that specifically binds to an antigen has a K value that is greater than when the molecule binds to another antigen. A K that is at least 2 logs, 2.5 logs, 3 logs, 4 logs, or more A Binding to an antigen. Binding can comprise preferential association of a binding motif, antibody, or antigen binding system with a target of the binding motif, antibody, or antigen binding system as compared to association of the binding motif, antibody, or antigen binding system with an entity that is not the target (i.e., a non-target). In some embodiments, the binding motif, antibody, or antigen binding system selectively binds to a target if the binding between the binding motif, antibody, or antigen binding system and the target is greater than 2-fold, greater than 5-fold, greater than 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or greater than 100-fold as compared to the binding between the binding motif, antibody, or antigen binding system and the non-target. In some embodiments, 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, then the binding motif, antibody or antigen binding system selectively binds to the target.

[0072] In another embodiment, the molecules that specifically bind to the antigen are present in an amount of about 1 x 10 -7 The dissociation constant (K d ) combination. In some embodiments, when K d About 1x10 -9 M to about 5x10 -9 M, the antigen binding molecule specifically binds the antigen with "high affinity". In some embodiments, when K d 1x10 -10 M to about 5x10 -10 M, the antigen-binding molecule specifically binds the antigen with "very high affinity". In one embodiment, the antigen-binding molecule has 10 -9 M of K dIn one embodiment, the dissociation rate is less than about 1×10 -5 In other embodiments, the antigen binding molecules are present in an amount of about 1×10 -7 M to about 1x10 -13 K between M d In yet another embodiment, the antigen binding molecule is present in an amount of about 1 x 10 -10 M to about 5x10 -10 M of K d Binds to human BCMA. In some embodiments, the antigen binding molecule is at about 1x10 -7 M and about 1x10 -13 K between M d In yet another embodiment, the antigen binding molecule is present in an amount of about 1x10 -10 M to about 5x10 -10 M of K d Binds to human CD19, CD20, or both.

[0073] In a specific embodiment, provided herein is an antibody or its antigen binding molecule, which binds to a target human antigen (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, provided herein is an antibody or its antigen binding molecule that binds to human CD19, human CD20, or both, with an affinity higher than another species of one or two target antigens, such as non-human CD19, non-human CD20, or both. In certain embodiments, provided herein is an antibody or its antigen binding molecule, which binds to a target human antigen (e.g., human BCMA or human CLL-1) with an affinity higher than another target antigen of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In certain embodiments, provided herein are antibodies or antigen binding molecules thereof that bind to human CD19, human CD20, or both with 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more affinity than another species of one or both target antigens, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assays. In specific embodiments, antibodies or antigen binding molecules thereof that bind to a target human antigen as described herein will bind to another target antigen with less than 10%, 15%, or 20% of the binding of the antibody or antigen binding molecule to the human antigen, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assays.

[0074] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to include a binding motif and a means of activating immune cells (e.g., T cells, such as naive T cells, central memory T cells, effector memory T cells, or a combination thereof) upon antigen binding. CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor. In some embodiments, CAR comprises 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 referred to as CAR T cells. "Extracellular domain" (or "ECD") refers to a portion of a polypeptide, which, when present in a cell membrane, is understood to be located in the extracellular space outside the cell membrane.

[0075] "Antigen" refers to any molecule that triggers an immune response or can be bound by an antibody or antigen binding molecule. An immune response may involve the production of antibodies or the activation of specific immunocompetent cells or both. Those skilled in the art will readily appreciate that any macromolecule (including nearly all proteins or peptides) can serve as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA or can be recombinantly expressed. Antigens can be specific to certain tissues, such as cancer cells, or they can be widely expressed. In addition, 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, an antigen binding system, or a binding agent (e.g., an antibody). In some embodiments, the target is an antigen or epitope of the present disclosure.

[0076] 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 effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or changes the binding ability of the ligand indirectly (e.g., the structure or energy of the ligand changes). In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof prevents the protein bound thereto from performing a biological function.

[0077] The term "autologous" refers to any material originating from the same individual into which it is later reintroduced. For example, the engineered autologous cell therapy (eACT) described herein TM ) involves collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, and then administering them back to the same patient.

[0078] The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species, such as an allogeneic T cell transplant.

[0079] The terms "transduction" and "transduced" refer to a process by which foreign 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 adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0080] "Transformation" refers to any process of introducing exogenous DNA into a host cell. Various methods can be used to transform under natural or artificial conditions. Any known method for inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell can be used to achieve transformation. In some embodiments, some transformation methods are selected based on the transformed host cell and / or the nucleic acid to be inserted. The transformation method may include but is not limited to viral infection, electroporation and lipofection. In some embodiments, the "transformed" cell is stably transformed because the inserted DNA can be replicated as an autonomously replicating plasmid or as a part of the host chromosome. In some embodiments, the transformed cell can express the introduced nucleic acid.

[0081] The term "vector" refers to an acceptor nucleic acid molecule modified to contain or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule, wherein additional DNA can be connected. Another type of vector is a viral vector, wherein additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. In addition, some vectors include sequences for guiding the expression of inserted genes, which are operably connected to these genes. Such vectors may be referred to as "expression vectors" herein. Standard techniques can be used for the engineering of vectors, for example, as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which are incorporated herein by reference for any purpose.

[0082] "Cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors. Examples of cancers that may be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods of the present disclosure can be used to reduce the size of tumors, such as tumors derived from the group consisting of bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach 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 intestine cancer, endocrine system cancer, thyroid cancer, thyroid gland cancer, Parathyroid cancer, adrenal cancer, 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), solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, cancers induced by the environment (including those induced by asbestos), other B cell malignancies and combinations of the cancers. In a specific embodiment, the cancer is multiple myeloma. Specific cancers may respond to chemotherapy or radiotherapy or the cancer may be refractory. Refractory cancer refers to cancer that is not amenable to surgical intervention and the cancer is either initially unresponsive to chemotherapy or radiation therapy, or the cancer becomes unresponsive over time. Cancer further includes relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma not otherwise specified (DLBCL), primary mediastinal large B-cell lymphoma after two or more lines of systemic therapy, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma.

[0083] As used herein, "anti-tumor effect" refers to a biological effect that can be presented as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various tumor-related physiological symptoms. Anti-tumor effect can also refer to the prevention of tumor occurrence, such as vaccines.

[0084] 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 immune responses. Cytokines can induce various responses in receptor cells. Cytokines can include steady-state cytokines, chemokines, proinflammatory cytokines, effectors and acute phase proteins. For example, steady-state cytokines (including interleukin (IL) 7 and IL-15) promote immune cell survival and proliferation, and proinflammatory cytokines can promote inflammatory responses. Examples of steady-state cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15 and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0085] "Chemokine" is a class of cytokines that mediate cell chemotaxis or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-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-induced protein 10 (IP-10), and thymus and activation-regulated chemokines (TARC or CCL17).

[0086] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent (e.g., engineered CAR T cells) is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to skilled practitioners, such as during clinical trials in human subjects, in animal model systems used to predict efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0087] The term "lymphocyte" includes natural killer (NK) cells, T cells or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocytes that represent the main components of the innate immune system. NK cells reject tumors and virus-infected cells. It works through the process of apoptosis or programmed cell death. Because it does not require activation to kill cells, it is called "natural killer". T cells play a major role in cell-mediated immunity (without antibody involvement). Its T cell receptor (TCR) distinguishes itself from other lymphocyte types. The thymus (a specialized organ of the immune system) is mainly responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (such as 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 Central memory T cells (i.e., naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express high levels of CD95, IL-2Rβ, CXCR3, and LFA-1, and display many of the unique functional attributes of memory cells; (ii) central memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express high levels of CD95, IL-2Rβ, CXCR3, and LFA-1, and display many of the unique functional attributes of memory cells; (iii) central memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express high levels of CD95, IL-2Rβ, CXCR3, and LFA-1, and display many of the unique functional attributes of memory cells; CM cells express L-selectin and CCR7, they secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory T EMCells, 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 the participation of antibodies). They produce antibodies and antigens and perform the role of antigen presenting cells (APCs), and transform into memory B cells after activation through antigen interaction. In mammals, immature B cells are formed in the bone marrow, which is where the name B cells come from.

[0088] "Linker" (L) or "linker domain" or "linker region" refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that connects any domain / region of the CAR of the present invention together. The linker may be composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other spatially, a longer linker can be used. 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 includes a picornavirus 2A-like linker, a CHYSEL sequence of porcine Teschovirus (P2A), a virus (T2A), or a combination, variant, and functional equivalent thereof. In other embodiments, the linker sequence may comprise Asp-Val / Ile-Glu-X-Asn-Pro-Gly. (2A) -Pro. (2B)Motif (SEQ ID NO:314), which results in cleavage between 2A glycine and 2B proline. Other joints are obvious to those skilled in the art and can be used in combination with alternative embodiments of the present invention. For example, in some embodiments, joints can be used to link or connect different antigen binding systems, such as two CARs of a bicistronic CAR. The joint can be part of a multi-element agent that connects different elements to each other. For example, a polypeptide comprising two or more functional domains or structural domains can include a stretch of amino acids that connect them to each other between such domains. In some embodiments, the polypeptide comprising a joint element has an overall 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 a joint. The joint can link or connect any domain / region of the CAR disclosed herein. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids in length (e.g., 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, or 10 to 100 amino acids in length). In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

[0089] "Single-chain variable fragment," "single-chain antibody variable fragment" or "scFv" antibody refers to an antibody format that comprises only the heavy and light chain variable regions, which are connected by a linker peptide.

[0090] The term "genetically engineered" or "engineered" refers to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte (e.g., a T cell), which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR) incorporated into the genome of the cell. Engineering generally includes artificial manipulation. For example, when two or more sequences that are not connected or linked together in this order in nature are artificially manipulated to be directly connected or linked to each other in an engineered polynucleotide, the polynucleotide is considered to be "engineered". In the case of operating cells by molecular biology techniques, if a cell or organism is manipulated so that its genetic information changes, the cell or organism is considered to be "engineered" (e.g., new genetic material that did not exist before is introduced, such as by transformation, somatic cell hybridization, transfection, transduction or other mechanisms, or changes or removal of previously existing genetic material, such as by substitution or deletion mutations, or by other schemes). In some embodiments, the binding agent is a modified lymphocyte, such as a T cell, which can be obtained from a patient or a 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 genome of the cell. The offspring of engineered polynucleotides or binding agents are generally referred to as "engineered", even if the actual operation is performed on a previous entity. In some embodiments, "engineered" refers to an entity that has been designed and produced. The term "designed" refers to an agent that (i) has an artificial structure or is artificially selected; (ii) it is produced by a process that requires artificiality; and / or (iii) it is different from natural substances and other known agents. "T cell receptor" or "TCR" refers to an antigen recognition molecule present on the surface of a T cell. During normal T cell development, each of the four TCR genes α, β, γ, and δ may be rearranged to produce highly diverse TCR proteins.

[0091] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Ab, cytokines, and complement) that results in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues in the body of a vertebrate.

[0092] The term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting a disease or recurrence of a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy may include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT) TM ) and allogeneic T cell transplantation. However, 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 Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and International Publication No. WO 2008 / 081035.

[0093] The T cells of immunotherapy can be from any source known in the art. For example, T cells can be differentiated from a hematopoietic stem cell population in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained using any number of techniques known to those skilled in the art (e.g., FICOLL TM Separation and / or apheresis) is obtained from a blood unit collected from a subject. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0094] The term "engineered autologous cell therapy" (which may be abbreviated as "eACT") is TM", also known as adoptive cell transfer) is the process of collecting a patient's own T cells and subsequently genetically altering them to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a specific tumor antigen, which is linked to an intracellular signaling portion comprising at least one co-stimulatory domain and at least one activation domain. The co-stimulatory domain can be derived from a naturally occurring co-stimulatory domain, such as one having the amino acid sequence of SEQ ID NO: 1 or a variant thereof (e.g., a variant having a truncated hinge domain ("THD")), and the activation domain can be derived from, for example, CD3-zeta. In certain embodiments, CARs are designed to have two, three, four or more co-stimulatory domains. CAR scFv can be designed to target, for example, CD19, which is a transmembrane protein expressed by cells of the B cell lineage (including all normal B cells and B cell malignancies, including but not limited to NHL, CLL, and non-T cell ALL). In some embodiments, CAR is engineered so that the costimulatory domain is expressed in the form of a single polypeptide chain. Example CAR T cell therapy and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference as a whole. "Adoptive cell therapy" or "ACT" involves transferring immune cells with anti-tumor activity to a subject, such as a cancer patient. In some embodiments, ACT is a method of treating a lymphocyte (e.g., engineered lymphocyte) with anti-tumor activity.

[0095] "Patient" includes any person suffering from cancer (eg, lymphoma or leukemia).Herein, the terms "subject" and "patient" are used interchangeably.

[0096] The term "in vitro" refers to events that occur in an artificial environment, such as in a test tube, reaction vessel, cell culture, etc., rather than in a multicellular organism. The term "in vitro cell" refers to any cell cultured in vitro. In particular, in vitro cells may include T cells. The term "in vivo" refers to events that occur in a multicellular organism, such as a human or non-human animal.

[0097] "Antigen-specific targeting region" (ASTR) refers to a CAR region that targets a specific antigen. The CAR of the present invention comprises at least two targeting regions targeting at least two different antigens. In one embodiment, CAR comprises three or more targeting regions targeting at least three or more different antigens. The targeting region on CAR is extracellular. In some embodiments, the antigen-specific targeting region comprises an antibody or its functional equivalent or its fragment or derivative thereof, and each targeting region targets a different antigen. The targeting region may comprise a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody or a double antibody, each of which is specific to the target antigen. However, there are many alternatives to promote immune responses, such as connected cytokines (which lead to recognition of cells with cytokine receptors), affinity bodies, ligand binding domains from naturally occurring receptors, soluble proteins / peptide ligands of receptors (e.g., on tumor cells), peptides and vaccines, each of which can be used in various embodiments of the present invention. In fact, as will be appreciated by those skilled in the art, almost any molecule that binds to a given antigen with high affinity can be used as an antigen-specific targeting region.

[0098] "Antigen presenting cell" or "APC" refers to a cell that processes and presents antigen 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.

[0099] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide 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. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as proteins, which 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.

[0100] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its associated ligand, wherein the binding mediates a signal transduction event. "Stimulatory molecule" refers to a molecule on a T cell, such as a T cell receptor (TCR) / CD3 complex that specifically binds to an associated stimulatory ligand present on an antigen presenting cell. "Stimulatory ligand" refers to a ligand that can specifically bind to a stimulatory molecule on a T cell when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), thereby mediating a primary response of the T cell (including but not limited to activation, initiation of an immune response, proliferation, etc.). Stimulatory ligands include but are not limited to anti-CD3 antibodies (e.g., OKT3), MHC class I molecules loaded with peptides, super agonist anti-CD2 antibodies, and super agonist anti-CD28 antibodies.

[0101] As used herein, a "co-stimulatory signal" refers to a signal that, when combined with a primary signal (eg, TCR / CD3 ligation), results in a T cell response (eg, but not limited to, proliferation and / or up- or down-regulation of key molecules).

[0102] As used herein, "costimulatory ligands" include molecules on antigen presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of costimulatory ligands provides signals that mediate T cell responses (including but not limited to proliferation, activation, differentiation, etc.). Costimulatory ligands induce signals in addition to primary signals provided by stimulatory molecules (e.g., provided by binding of T cell receptor (TCR) / CD3 complexes to major histocompatibility complex (MHC) molecules loaded with peptides). 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, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible co-stimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2 or programmed death (PD) L1. Co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds 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).

[0103] "Co-stimulatory molecules" are cognate binding partners that specifically bind to co-stimulatory ligands on T cells, thereby mediating the co-stimulatory response of T cells (such as but not limited to proliferation). Co-stimulatory molecules include but are not limited to 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45. CD100(SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160(BY55), CD18, CD19, CD19a, CD2, C D22, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3(alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD8 6. 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, ITGBl, 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 (CDl la / CD18), MHC Class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof.

[0104] Herein, the terms "reduce" and "reduction" are used interchangeably and refer to any change that is less than the original. "Reduce" and "reduction" are relative terms that require a comparison between before and after the measurement. "Reduce" and "reduction" include complete depletion.

[0105] The terms "improve," "increase," "inhibit," and "decrease" refer to values ​​relative to a baseline or other reference measurement. In some embodiments, appropriate reference measurements may include measurements in certain systems (e.g., a single individual) under otherwise comparable conditions in the absence of an agent or treatment (e.g., before and / or after), or in the presence of an appropriate comparable reference agent. In some embodiments, appropriate reference measurements may include measurements in comparable systems that are known or expected to respond in a comparable manner in the presence of a relevant agent or treatment.

[0106] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on the subject, or the subject is administered an active agent to achieve the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or illnesses or biochemical indicators associated with the disease. In one embodiment, "treatment" or "treatment" includes partial relief. In another embodiment, "treatment" or "treatment" includes complete relief. In some embodiments, treatment may be directed to subjects who do not show signs of related diseases, illnesses and / or conditions and / or subjects who only show early signs of diseases, illnesses and / or conditions. In some embodiments, such treatment may be directed to subjects who show one or more confirmed signs of related diseases, illnesses and / or conditions. In some embodiments, treatment may be directed to subjects who have been diagnosed with related diseases, illnesses and / or conditions. In some embodiments, treatment may be directed to subjects who are known to have one or more susceptibility factors statistically associated with related diseases, illnesses and / or conditions.

[0107] The term "agent" may refer to any class of molecules or entities comprising the following, or to a plurality of molecules or entities of which any one may be used as the following: 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 agent may be used in an isolated or pure form. In some embodiments, the agent may be used in a crude or impure form. In some embodiments, the agent may be provided as a colony, collection or library, for example, which may be screened to identify or characterize members present therein.

[0108] Two events or entities are "associated" with each other if the presence, level, and / or form of one event or entity is associated with the other. For example, an entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is considered to be associated with a disease, disorder, or condition if the presence, level, and / or form of the entity is associated with the occurrence and / or susceptibility to the disease, disorder, or condition (e.g., in a relevant population). For example, if two or more entities interact directly or indirectly so that they are in physical proximity and / or remain in physical proximity (e.g., bound) to each other, they are physically "associated" with each other. In other examples, two or more entities that are physically associated with each other are covalently linked or linked to each other, or non-covalently associated, such as by hydrogen bonds, van der Waals interactions, hydrophobic interactions, attractive forces, and combinations thereof.

[0109] The term "identity" refers to the overall correlation between polymeric molecules, such as nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules. The method for calculating the percentage identity between two provided polypeptide sequences is known. The calculation of the percentage identity of two nucleic acids or polypeptide sequences, for example, can be carried out by comparing two sequences for the best comparison purpose (for example, a room can be introduced in one or both of the first and second sequences to perform the best comparison, and for the purpose of comparison, different sequences can be ignored). Then the nucleotides or amino acids at the corresponding positions are compared. When a position in the first sequence is occupied by the same residue (such as nucleotides or amino acids) as the corresponding position in the second sequence, the molecules are the same at this position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally considering the number of rooms and the length of each room (which may need to be introduced to achieve the best comparison of the two sequences). The comparison or comparison of sequences and the determination of the percentage identity between the two sequences can be completed using a mathematical algorithm, such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are 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%).

[0110] To calculate percent identity, the compared sequences are usually aligned in a manner that produces the largest match between the sequences. An example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides are optimally matched (such as the "matched span" determined by the algorithm). In certain embodiments, the algorithm also uses a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352, for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 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, Gap BLAST, and PSI-BLAST for amino acid sequences.Exemplary such programs 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 Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying similar sequences, the above programs typically provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be substantially similar if, over a corresponding stretch of residues (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%), at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the corresponding residues of the two sequences are similar and / or identical. In some embodiments, the corresponding stretch is the entire sequence. In some embodiments, the corresponding stretch is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues. Sequences with substantial sequence similarity may be homologs of each other.

[0111] "Combination therapy" refers to a situation in which a subject is exposed to two or more treatment regimens (e.g., two or more treatment moieties) at the same time. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may involve administering one or more agents or modalities to a subject who is receiving other agents or modalities in the combination. For clarity, combination therapy does not require that the separate agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents or their active portions may be used together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0112] "Corresponding to" can be used to designate the position / identity of a structural element in a molecule or composition by comparison to an appropriate reference molecule or composition. For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be identified as "corresponding to" a residue in an appropriate reference polymer. For example, for simplicity, residues in a polypeptide can be designated using a canonical numbering system based on a reference related polypeptide, such that the amino acid "corresponding to" the residue at position 100, for example, does not actually have to be the 100th amino acid in the amino acid chain, as long as it corresponds to the residue found at position 100 in the reference polypeptide. Various sequence alignment strategies are available, including software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, 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 polypeptides and / or nucleic acids according to the present disclosure.

[0113] The term "domain" refers to a portion of an entity. In some embodiments, a "domain" is associated with a structural and / or functional characteristic of an entity, for example, such that when the domain is physically separated from the rest of its parent entity, it substantially or completely retains the structural and / or functional characteristic. In some embodiments, a domain may comprise a portion of an entity that, when separated from the (parent) entity and connected or linked to a different (recipient) entity, substantially retains and / or confers one or more structural and / or functional characteristics to the recipient entity, for example, its characteristics in the parent entity. In some embodiments, a domain is part of a molecule (e.g., a small molecule, a carbohydrate, a lipid, a nucleic acid, or a polypeptide). In some embodiments, a domain is part of a polypeptide; in some such embodiments, a domain is characterized by a structural element (e.g., an amino acid sequence or sequence motif, an alpha helical characteristic, a beta folding characteristic, a coiled coil characteristic, a random coil characteristic, etc.), and / or by a functional characteristic (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0114] 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 an antibody) for administration to a subject. Typically, each such unit contains a predetermined amount of active agent. In some embodiments, such an amount is an amount (or all fractions thereof) of a unit dose suitable for administration according to a dosing regimen, which, when administered to a relevant population, has been determined to be associated with a desired or beneficial outcome. The total amount of the therapeutic composition or medicament administered to a subject is determined by one or more practitioners and may involve the administration of more than one dosage form.

[0115] The term "dosage regimen" can be used to refer to a group 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 separated from the other doses in time. In some embodiments, the dosing regimen comprises multiple doses and consecutive doses are separated from each other by time periods of equal length; in some embodiments, the dosing regimen comprises multiple doses and consecutive doses are separated from each other by at least two time periods of different lengths. In some embodiments, all doses within the dosing regimen have the same amount of unit dose. In some embodiments, different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose having the amount of a first dose, followed by one or more additional doses having the amount of a second dose different from the amount of the first dose. In some embodiments, the dosing regimen is adjusted regularly to achieve desired or beneficial results.

[0116] "Effect function" refers to the biological result of the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector function includes, but is not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector function can be dependent on, independent of, or both antigen binding. ADCC refers to the lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve effector cells carrying Fc receptors (FcRs) that recognize and subsequently kill antibody-coated target cells (e.g., cells expressing antibody-bound antigens on their surfaces). Effector cells mediating ADCC may include immune cells, including, but not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0117] "Effector cells" refer to cells of the immune system 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, 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 of any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0118] The term "excipient" refers to an agent that can be included in the composition, for example, to provide or contribute to the desired consistency or stabilizing effect. In some embodiments, suitable excipients can include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.

[0119] A "fragment" or "portion" of a material or entity as described herein has a structure comprising discrete portions of a whole, such as discrete portions of a physical entity or an abstract entity. In some embodiments, a fragment lacks one or more portions found in the whole. In some embodiments, a fragment consists of or comprises characteristic structural elements, domains, or portions found in the whole. In some embodiments, a polymer fragment comprises, or consists of, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., residues) as found in the overall polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) as found in the overall polymer (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the overall material or entity may be referred to as the "parent" of the fragment.

[0120] The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. Generally, if the two segments are (1) portions that are not naturally contained in the same peptide, and / or (2) portions that have not been previously linked or connected in a single polypeptide, and / or (3) portions that are linked or connected to each other by artificial action, then a polypeptide containing at least two such segments is considered to be a fusion polypeptide.

[0121] The term "gene product" or "expression product" generally refers to RNA transcribed from a gene (before and / or after processing) or a polypeptide encoded by RNA transcribed from a gene (before and / or after modification).

[0122] The term "isolated" refers to a substance that (1) has been separated from at least some components with which it was associated at an earlier time or with which it would otherwise be associated, and / or (2) is present in a composition that contains limited or defined amounts or concentrations of one or more known or unknown contaminants. In some embodiments, an isolated substance can 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-substance components (which were associated with the substance at an earlier time, such as other components or contaminants with which the substance was previously or will be otherwise associated). In some cases, a substance is isolated if it is present in a composition containing limited or reduced amounts or concentrations of the same or similar type of molecules. For example, in some cases, if nucleic acid, DNA or RNA material is present in a composition comprising a limited or reduced amount or concentration of non-material nucleic acid, DNA or RNA molecule, the nucleic acid, DNA or RNA material is separated. For example, in some cases, if polypeptide material is present in a composition comprising a limited or reduced amount or concentration of non-material polypeptide molecules, the polypeptide material is separated. In certain embodiments, amount can be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, limited amount can be an amount that is no more than 100% of the amount of the substance in the composition, for example, no more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) of the amount of the substance in the composition. In some cases, the composition is pure or substantially pure for the selected substance. In some embodiments, the isolated 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%). A substance is "pure" if it is substantially free of other components or free of contaminants. In some embodiments, a substance may still be considered "isolated" or even "pure" after 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 separation percentage or purity of the substance is calculated without including such carriers or excipients.

[0123] "Nucleic acid" refers to any polymeric chain of nucleotides. The nucleic acid can be DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, the nucleic acid comprises one or more nucleic acid analogs. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) based on polymerization of a complementary template, 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, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length (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 that comprises at least one element encoding a polypeptide or is the complement of a sequence encoding a polypeptide.

[0124] "Operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, a control element "operably linked" to a functional element is associated in a manner that achieves expression and / or activity of the functional element under conditions compatible with the control element.

[0125] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to the recipient, or the benefits to the recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate compositions as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not harmful to the recipient, or the benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in carrying or transporting a medicament from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or the benefits to the recipient must outweigh any harmful 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth 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 nontoxic, compatible substances used in pharmaceutical formulations.

[0126] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant subject or population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to forms suitable for the following: 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, for example, a sterile solution or suspension, or a sustained release formulation; topical application, such as application to the skin, lungs, or mouth as a cream, ointment, or controlled release patch or spray; intravaginal or intrarectal, such as as a pessary, cream or foam; sublingual; ophthalmic; transdermal; or nasal, lungs, and application to other mucosal surfaces.

[0127] The term "reference" describes a standard or control with which it is compared. For example, in some embodiments, an agent, animal, individual, colony, sample, sequence or value of interest is compared with a reference or control as an agent, animal, individual, colony, 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, a reference or control is determined or characterized under conditions or environments comparable to the subject of evaluation. When there is enough similarity to justify reliance and / or comparison on a selected reference or control.

[0128] "Regulatory T cells" ("Treg", "Treg cells" or "Tregs") refer to a CD4+T lymphocyte lineage that is involved in controlling certain immune activities (e.g., autoimmunity, allergic reactions, and responses to infection). Regulatory T cells can regulate the activity of T cell populations and can also affect certain innate immune system cell types. Tregs can be identified by the expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically account for 5-10% of peripheral CD4+T lymphocytes. However, in the Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells), Treg cells may account for up to 20-30% of the total CD4+T lymphocyte population.

[0129] The term "sample" generally refers to an aliquot of a material obtained or derived from a source of interest. In some embodiments, the source of interest is biological or environmental origin. In some embodiments, the source of interest may include cells or organisms, such as cell mass, tissue or animal (e.g., people). In some embodiments, the source of interest includes biological tissue or fluid. In some embodiments, biological tissue or fluid may include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculatory fluid, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheumatism, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomitus and / or its combination or component. In some embodiments, biological fluid may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph and / or transcellular fluid. In some embodiments, biological fluid can comprise plant exudate.In some embodiments, biological tissue or sample can be obtained by, for example, suction, biopsy (for example, fine needle or tissue biopsy), swab (such as oral, nasal, skin or vaginal swab), scraping, surgery, washing or lavage (for example, bronchoalveolar, duct, nose, eye, mouth, uterus, vagina or other flushing or lavage).In some embodiments, biological sample comprises the cell obtained from individual.In some embodiments, sample is " primary sample " obtained directly from the source of interest by any suitable means.In some embodiments, it will be clear from the context that the term " sample " refers to the preparation obtained by processing (for example, by removing one or more components and / or by adding one or more medicaments thereto) primary sample.Such " processed sample " can comprise nucleic acid or protein obtained, for example, from sample extraction or by subjecting primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, separation and / or purification of certain components, etc.

[0130] The term "cancer stage" refers to a qualitative or quantitative assessment of the level of cancer progression. In some embodiments, the criteria for determining the cancer stage may include, but are not limited to, one or more of the location of the cancer in the body, the size of the tumor, whether the cancer has spread to the lymph nodes, whether the cancer has spread to one or more different parts of the body, etc. In some embodiments, the so-called TNM system can be used to stage cancer, according to which T refers to the size and extent of the main tumor (commonly referred to as the primary tumor); N refers to the number of lymph nodes with cancer nearby; and M refers to whether the cancer has metastasized. In some embodiments, 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 cancer), stage I-III (cancer is present; the larger the number, the larger the tumor, and the higher the degree of spread to nearby tissues) or stage IV (cancer spreads to distant parts of the body). In some embodiments, a cancer can be assigned to a stage selected from the group consisting of: in situ; localized (the cancer is limited to where it started, with no signs of spreading); 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).

[0131] The phrase "therapeutic agent" may refer to any agent that causes a desired pharmacological effect when applied to an organism. In some embodiments, if the agent shows a statistically significant effect in a suitable population, the agent is considered to be a therapeutic agent. In some embodiments, a suitable population may be a population of a model organism or a human subject. In some embodiments, a suitable population may be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical condition, according to the presence or absence of a biomarker, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, alleviate, inhibit, prevent one or more symptoms or features of a disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce its incidence. In some embodiments, a therapeutic agent is an agent that has been or needs to be approved by a government agency before it can be marketed for application to humans. In some embodiments, a therapeutic agent is an agent that requires a medical prescription to be applied to humans.

[0132] Various aspects of the present disclosure are further described in detail in the following subsections. The present disclosure provides antigen binding systems and binding agents comprising at least an anti-CD20 binding motif. Among them, the present disclosure provides methods and compositions for treating cancer and / or for initiating or regulating an immune response. In certain embodiments, the present disclosure comprises dual-targeted antigen binding systems and binding agents because they comprise an anti-CD20 binding motif and a second binding motif for 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 the present disclosure is a chimeric antigen receptor. In some embodiments, the antigen binding system of the present disclosure is a bispecific or bicistronic chimeric antigen receptor. In some embodiments, the binding agent of the present disclosure is an engineered T cell receptor.

[0133] Various embodiments of the present disclosure provide vectors encoding binding motifs or antigen binding systems provided herein, e.g., vectors encoding anti-CD20 / anti-CD19 antigen binding systems, such as bispecific or bicistronic anti-CD20 / anti-CD19 chimeric antigen receptors. Various embodiments of the present disclosure provide binding agents, which are cells encoding or expressing antigen binding systems or binding motifs provided herein, e.g., 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. The present disclosure provides binding agents, e.g., comprising binding agents as genetically modified immune cells with integrated genes, e.g., nucleotide sequences of interest (e.g., constitutive expression constructs and / or inducible expression constructs comprising such nucleotide sequences). In some embodiments, the present disclosure provides methods for treating subjects with tumors, comprising administering to the subject a binding agent therapy described herein and / or a protein therapeutic agent described herein. In some embodiments, the method further comprises administering one or more additional therapies (e.g., a second binding agent described herein (e.g., CAR-T cells, CAR-NK cells, TCR-T cells, TIL cells, allogeneic NK cells, and autologous NK cells)), an antibody-drug conjugate, an antibody, a bispecific antibody, a T cell engaging bispecific antibody, an engineered antibody, and / or a polypeptide).

[0134] Other features, objects and advantages of the present disclosure will be apparent in the following detailed description.It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only and not limitation.

[0135] The anti-CD20 binding motifs of the present disclosure may comprise antigen binding sequences as found in antibodies described herein. In some cases, the anti-CD20 binding motifs of the present disclosure comprise antigen binding fragments described herein. Unless otherwise indicated, it is understood that the reference to CD20 in the present disclosure relates to human CD20. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one heavy chain CDR (HCDR) provided herein, such as at least one HCDR disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two HCDRs provided herein, such as at least two HCDRs disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three HCDRs provided herein, such as three HCDRs disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one light chain CDR (LCDR) provided herein, such as at least one LCDR disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two LCDRs provided herein, e.g., at least two LCDRs disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three LCDRs provided herein, e.g., three LCDRs disclosed in any one of Tables 4-13.

[0136] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one HCDR provided herein, e.g., at least one HCDR disclosed in any one of Tables 4-13, and at least one LCDR provided herein, e.g., at least one LCDR disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise one HCDR provided herein, e.g., at least one HCDR disclosed in any one of Tables 4-13, and one LCDR provided herein, e.g., derived from the same table in Table 4-13 as the HCDR. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in any one of Tables 4-13, and two LCDRs provided herein, e.g., at least two LCDRs disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in any one of Tables 4-13, and two LCDRs provided herein, e.g., derived from the same table in Table 4-13 as the HCDR. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in any one of Tables 4-13, and three LCDRs provided herein, e.g., three LCDRs disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in any one of Tables 4-13, and three LCDRs derived from the same table in Tables 4-13 as the HCDRs.

[0137] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one heavy chain framework region (heavy chain FR) of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR chain of a heavy chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13.

[0138] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13.

[0139] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in any one of Tables 4-13, and at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in any one of Tables 4-13, and one light chain FR of a light chain variable domain disclosed herein, e.g., derived from the same table in Tables 4-13 as the heavy chain FR. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two heavy chain FRs of a heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13, and two light chain FRs of a light chain variable domain disclosed herein, such as at least two light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two heavy chain FRs of a heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13, and two light chain FRs of a light chain variable domain disclosed herein, such as derived from the same table in Tables 4-13 as the heavy chain FRs. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13, and three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13, and three light chain FRs derived from the same table in Tables 4-13 as the heavy chain FRs.

[0140] The exemplary antibody sequences provided in Tables 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 provided in Tables 4-13 and portions thereof may be included in the binding motif.

[0141] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise one, two or three FRs, which together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95% or 100%, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) with the FRs of the corresponding heavy chain variable domains of the heavy chain variable domains disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise one, two or three FRs, which together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95% or 100%) with the FRs of the corresponding light chain variable domains of the light chain variable domains disclosed in any one of Tables 4-13.

[0142] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two heavy chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of Tables 4-13, which heavy chain variable domains may be the same or different.

[0143] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two light chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of Tables 4-13, which light chain variable domains may be the same or different.

[0144] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of Tables 4-13, and at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise a heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of Tables 4-13, and a heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of Tables 4-13. The light chain variable domain disclosed in any one of Tables 4-13 has a light chain variable domain with 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 domain and light chain variable domain are optionally derived from the same table in Tables 4-13.

[0145] In various embodiments, the anti-CD20 binding motifs of the present disclosure comprise two heavy chain variable domains, each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in any one of Tables 4-13, and two light chain variable domains, each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in any one of Tables 4-13. 0%, at least 95% or 100% identity; for example 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) light chain variable domains, wherein in various embodiments, (i) each heavy chain variable domain can be the same or different; (ii) each light chain variable domain can be the same or different; (iii) at least one heavy chain variable domain and at least one light chain variable domain can be derived from the same table in Tables 4-13; or (iv) two heavy chain variable domains and two light chain variable domains are derived from the same table in Tables 4-13. Each of Tables 4-13 represents a heavy chain variable domain and a light chain variable domain sequence of an exemplary antibody, which comprises (i) a heavy chain variable domain of an exemplary antibody; (ii) a DNA sequence encoding a heavy chain variable domain; (iii) three heavy chain variable domain CDRs of the heavy chain variable domain numbered according to IMGT, Kabat, and Chothia; (iv) a light chain variable domain of an exemplary antibody; (v) a DNA sequence encoding a light chain variable domain; and (vi) three light chain variable domain CDRs of the light chain variable domain numbered according to IMGT, Kabat, and Chothia. The information provided in each table provides a framework amino acid sequence, as well as a nucleotide sequence encoding each CDR amino acid sequence and a nucleotide sequence encoding a corresponding FR amino acid sequence.

[0146] In various embodiments, the binding motif can comprise a heavy chain variable domain of the present disclosure (e.g., one having at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95% or 100% identity to a heavy chain variable domain in any one of Tables 4-13; 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., one having at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95% or 100% identity to a light chain variable domain in any one of Tables 4-13; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%), and a linker (e.g., a linker according to SEQ ID NO: 247 and / or a linker according to SEQ ID NO: 248. NO:307-313; see, e.g., Whitlow et al. Protein Eng. 1993 Nov; 6(8):989-95). In various embodiments, the binding motif can comprise a leader sequence, a heavy chain variable domain of the present disclosure (e.g., one that has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95% or 100% identity to a heavy chain variable domain in any one of Tables 4-13; 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., one that has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95% or 100% identity to a light chain variable domain in any one of Tables 4-13; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%), and a linker. If the amino acid or nucleotide sequence of the binding motif comprising the heavy chain variable domain of the present disclosure and the light chain variable domain of the present disclosure is provided, the joint of the two variable domains will be obvious from the sequence based on the present disclosure. If the amino acid or nucleotide sequence of the binding motif comprising the heavy chain variable domain of the present disclosure and the light chain variable domain of the present disclosure is provided, the leader sequence will be obvious according to the present disclosure. For the avoidance of doubt, the heavy chain variable domain and the light chain variable domain of the present invention can exist in any direction, such as the direction in which the heavy chain variable domain is at the C-terminus of the light chain variable domain or the N-terminus of the heavy chain variable domain. In various embodiments, the binding motif may include a joint according to SEQ ID NO:247 adjacent to one or more other joints and / or a joint according to any one of SEQ ID NO:307-313.

[0147] In certain embodiments, the anti-CD20 binding motifs of the present disclosure comprise a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity to 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 certain embodiments, the anti-CD20 binding motifs of the present disclosure comprise a binding motif comprising a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307-313. In certain embodiments, the anti-CD20 binding motifs of the present disclosure comprise a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity to 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 certain embodiments, the anti-CD20 binding motifs of the present disclosure comprise a binding motif comprising a CSF2RA leader sequence according to SEQ ID NO: 245. In certain embodiments, the anti-CD20 binding motifs of the present disclosure comprise a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, a linker of the present disclosure, and a leader sequence of the present disclosure. Exemplary nucleotide sequences encoding anti-CD19 binding motifs and components thereof are found in SEQ ID NOs: 246 and 248. In various embodiments, the binding motifs of the present disclosure have a sequence according to any one of the sequences of Table 53 (SEQ ID NOs: 251-260).

[0148] Binding agents of the present disclosure based on the exemplary antibodies provided herein (such as, for example, Ab1) can be provided in any fragment or form comprising a heavy chain variable domain according to the exemplary antibodies shown and a light chain variable domain according to the exemplary antibodies shown.

[0149] Table 4: Exemplary Antibody Sequence 1 (Ab1)

[0150]

[0151]

[0152] Table 5: Exemplary antibody sequence 2 (Ab2)

[0153]

[0154]

[0155]

[0156] Table 6: Exemplary antibody sequence 3 (Ab3)

[0157]

[0158]

[0159]

[0160] Table 7: Exemplary Antibody Sequence 4 (Ab4)

[0161]

[0162]

[0163] Table 8: Exemplary Antibody Sequence 5 (Ab5)

[0164]

[0165]

[0166]

[0167] Table 9: Exemplary antibody sequence 6 (Ab6)

[0168]

[0169]

[0170] Table 10: Exemplary antibody sequence 7 (Ab7)

[0171]

[0172]

[0173] Table 11: Exemplary Antibody Sequence 8 (Ab8)

[0174]

[0175]

[0176]

[0177] Table 12: Exemplary Antibody Sequence 9 (Ab9)

[0178]

[0179]

[0180] Table 13: Exemplary Antibody Sequence 10 (Ab10)

[0181]

[0182]

[0183] The present disclosure includes antibodies and antigen binding systems, which include 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)). The dual-targeted antigen binding system includes a bispecific CAR and a bicistronic CAR. Many antigen binding motifs are known. In various embodiments, the second target antigen is CD19. The present description includes a variety of second 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, combined HER1-HER2, combined HER2-HER3, HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, IL-11Ralpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate-specific antigen, ROR1 or VEGFR2, or a combination thereof. Thus, in various embodiments, the antigen binding system or antibody of the present disclosure may comprise a first binding motif that is an anti-CD20 binding motif and a first binding motif that is a binding motif for 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 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 gpl20, IL-11Ralpha, kappa chain, lambda chain, melanoma associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate specific antigen, ROR1 or VEGFR2 or a second binding motif for a second antigen of a combination thereof. In some embodiments, the second antigen is a characteristic antigen of a B cell or a subset thereof, optionally wherein the second antigen is not CD19 or CD20. Examples of binding motifs targeting these second antigens are known and / or provided herein.

[0184] In some cases, in an antigen binding system such as a bispecific CAR comprising an anti-CD20 binding motif (e.g., comprising a heavy chain variable domain and / or a light chain variable domain of the present disclosure) and an anti-CD19 binding motif (e.g., comprising a heavy chain variable domain and / or a light chain variable domain of the present 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 than its heavy chain variable domain and / or light chain variable domain). In some cases, in an antigen binding system such as a bispecific CAR comprising an anti-CD20 binding motif (e.g., comprising a heavy chain variable domain and / or a light chain variable domain of the present disclosure) and an anti-CD19 binding motif (e.g., comprising a heavy chain variable domain and / or a light chain variable domain of the present 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 agent than the anti-CD19 binding motif (or than its heavy chain variable domain and / or light chain variable domain).

[0185] 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 indicated, it should be understood that the reference to CD19 in the present disclosure relates to human CD19. It is found on the surface of B cells. Since CD19 expresses a marker of B cells, it can be used as an antigen, such as identifying B cells and cancer cells derived from B cells, such as B cell lymphomas. Anti-CD19 antibodies can bind to CD19 expressed in, for example, B lymphocytes, B cell chronic lymphocytic leukemia (B-CLL) cells, prolymphocytic leukemia (PLL) cells, hairy cell leukemia (HCL) cells, common acute lymphoblastic leukemia (CALL) cells, pre-B acute lymphoblastic leukemia (pre-B-ALL) cells, and NULL acute lymphoblastic leukemia (NULL-ALL) cells in peripheral blood and spleen, to provide some non-limiting examples. An exemplary pharmaceutical product comprising an antigen binding system comprising an anti-CD19 binding motif is a pharmaceutical product It is a CD19-directed, genetically modified autologous T-cell immunotherapy indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma (see FDA-approved package insert for methods and compositions related to immunotherapy, the entire contents of which are incorporated herein by reference). Another exemplary drug product comprising an antigen binding system comprising an anti-CD19 binding motif is a drug product It is a CD19-directed, genetically modified autologous T cell immunotherapy indicated for the treatment of: (1) patients 25 years of age or younger with refractory or second or later relapsed B-cell precursor acute lymphoblastic leukemia (ALL); and (2) adult patients with relapsed or refractory (r / r) large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma (see FDA-approved package insert for methods and compositions related to immunotherapy, the entire contents of which are incorporated herein by reference).

[0186] and Both contain antibody binding domains derived from anti-human CD19 antibodies. Many anti-CD19 antibodies are thought to bind to an epitope of CD19 encoded in exon 4 of the CD19 gene. Other anti-CD19 binding motifs can recognize different epitopes of CD19, or the same epitope with different affinities. The antigen binding system can include an antigen binding domain derived from, for example, SJ25C1. CD19 antibody, clone SJ25C1 is derived from 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.

[0187] The anti-CD19 binding motifs of the present disclosure may comprise antigen binding sequences as found in antibodies described herein. In some embodiments, the anti-CD19 binding motifs of the present disclosure comprise antigen binding fragments provided herein.

[0188] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one HCDR provided herein, e.g., at least one HCDR disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in Table 14.

[0189] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one LCDR provided herein, e.g., at least one LCDR disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two LCDRs provided herein, e.g., at least two LCDRs disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise three LCDRs provided herein, e.g., three LCDRs disclosed in Table 14.

[0190] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one HCDR provided herein, e.g., at least one HCDR disclosed in Table 14, and at least one LCDR provided herein, e.g., at least one LCDR disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two HCDRs provided herein, e.g., at least two HCDRs disclosed in Table 14, and two LCDRs provided herein, e.g., at least two LCDRs disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise three HCDRs provided herein, e.g., three HCDRs disclosed in Table 14, and three LCDRs provided herein, e.g., three LCDRs disclosed in Table 14.

[0191] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one heavy chain framework region (heavy chain FR) of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR chain of a heavy chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in Table 14.

[0192] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in Table 14.

[0193] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise 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 Table 14, 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 Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two heavy chain FRs of a heavy chain variable domain disclosed herein, such as at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 14, and two light chain FRs of a light chain variable domain disclosed herein, 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 motifs of the present disclosure comprise three heavy chain FRs of a heavy chain variable domain disclosed herein, such as three heavy chain FRs of a heavy chain variable domain disclosed in Table 14, and three light chain FRs of a light chain variable domain disclosed herein, such as three light chain FRs of a light chain variable domain disclosed in Table 14.

[0194] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise one, two or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95% or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) with the FRs of the corresponding heavy chain variable domains disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise one, two or three FRs that together or each individually have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95% or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) with the FRs of the corresponding light chain variable domains disclosed in Table 14.

[0195] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two heavy chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the heavy chain variable domains disclosed in Table 14, which heavy chain variable domains may be the same or different.

[0196] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in Table 14. In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two light chain variable domains, each of which has at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the light chain variable domains disclosed in Table 14, which light chain variable domains may be the same or different.

[0197] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a heavy chain variable domain disclosed in Table 14, and at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a light chain variable domain disclosed in Table 14.

[0198] In various embodiments, the anti-CD19 binding motifs of the present disclosure comprise two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to 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%) to the light chain variable domains disclosed in Table 14. The variable domains have a light chain variable domain with 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.

[0199] In certain embodiments, the anti-CD19 binding motifs of the present disclosure comprise a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 247. In certain embodiments, the anti-CD19 binding motifs of the present disclosure comprise a binding motif comprising a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307-313. In certain embodiments, the anti-CD19 binding motifs of the present disclosure comprise a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) with SEQ ID NO: 245. In certain embodiments, the anti-CD19 binding motifs of the present disclosure comprise a binding motif comprising a CSF2RA leader sequence according to SEQ ID NO: 245. In certain embodiments, the anti-CD19 binding motifs of the present disclosure comprise a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, a linker of the present disclosure, and a leader sequence of the present disclosure. In certain embodiments, the binding motif has a sequence as shown in SEQ ID NO: 243. Exemplary nucleotide sequences encoding anti-CD19 binding motifs and components thereof are found in SEQ ID NOs: 244, 246 and 248. In various embodiments, the binding motif may comprise a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307-313, adjacent to one or more additional linkers.

[0200] Table 14: Exemplary anti-CD19 antibody sequences (Ab11)

[0201]

[0202]

[0203]

[0204] Antigen binding system includes, for example, bispecific and bicistronic chimeric antigen receptors (CAR). The present disclosure particularly provides an antigen binding system targeting CD20 and a second target antigen such as CD19. In some embodiments, the antigen binding system of the present disclosure includes a bispecific antigen binding system. In some embodiments, the antigen binding system of the present disclosure includes a bicistronic antigen binding system (e.g., a system comprising a first CAR and a second CAR, wherein the first and second CARs are expressed in the same cell). Bicistronic CARs may include two CARs that bind to different targets and are encoded by a single vector. Bicistronic CARs may include a first CAR comprising an anti-CD19 binding motif and a second CAR comprising an anti-CD20 binding motif. The binding motifs associated with various CAR frameworks are interchangeable, and the combination of features provided in this embodiment is exemplary and non-restrictive. In some embodiments, the first CAR and the second CAR of bicistronic CARs (e.g., anti-CD20 CARs and anti-CD19 CARs) are encoded by separate genes and / or expressed as separate mRNA molecules. In some embodiments, the first CAR and the second CAR 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 a first CAR, a cleavable linker domain, and a 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).

[0205] In various embodiments, the bicistronic CAR vector utilizes a ribosome skipping sequence or an internal ribosome entry site. A single vector encoding two independent CAR molecules separated by a ribosome skipping sequence can express a bicistronic CAR. In various embodiments, the bicistronic CAR includes a first CAR and a second CAR, wherein the sequence of the first CAR and the second CAR is different only in terms of binding motifs. In various embodiments, the bicistronic CAR includes a first CAR and a second CAR, wherein the sequence of the first CAR and the second CAR is different only in terms of heavy chain variable domain sequence and / or light chain variable domain sequence. Therefore, in some embodiments, the first CAR and the second CAR of the bicistronic CAR can have the same or different sequences for any one or all components (such as the same or different costimulatory domains) of one or more components thereof. For example, one or both of the first CAR and the second CAR of the bicistronic CAR can include a costimulatory domain provided herein, such as CD28, 41BB, OX40 or ICOS costimulatory domains.

[0206] The CAR of the bicistronic CAR may include a binding motif, a hinge, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The binding motif may be an anti-CD19 or anti-CD20 binding motif disclosed herein. The hinge and transmembrane domain may be a 28T (CD28) domain or a CD8K domain comprising a hinge domain and a transmembrane domain. The costimulatory domain may be a CD28 or 41BB costimulatory domain. The activation domain may be a CD3z activation domain.

[0207] In some embodiments, both the first binding motif and the second binding motif (e.g., different anti-CD20 and anti-CD19 binding motifs) are included in a single bispecific CAR. In such bispecific CARs, the CAR molecule itself can be engineered to recognize more than one antigen. In a tandem bispecific CAR, the first and second binding motifs are extracellular and can be characterized as a membrane proximal binding motif and a membrane distal binding motif. In some embodiments, the anti-CD20 binding motif is membrane proximal, and the anti-CD19 binding motif is membrane distal. In other embodiments, the anti-CD19 binding motif is membrane distal, and the anti-CD20 binding motif is membrane proximal.

[0208] Chimeric antigen receptors (CARs) are engineered receptors that can guide or redirect T cells (e.g., patient or donor T cells) to target selected antigens. CARs can be engineered to recognize antigens and activate immune cells to attack and destroy cells with the antigen when bound to the antigen. When these antigens are present on tumor cells, immune cells expressing CARs can target and kill tumor cells. CARs typically include extracellular binding motifs (e.g., anti-CD20 and / or anti-CD19 binding motifs) that mediate antigen binding, when the antigen binding system is present on the cell surface or at the cell membrane, a transmembrane domain that spans or is understood to span the cell membrane, and an intracellular (or cytoplasmic) signaling domain.

[0209] According to at least one non-limiting viewpoint, there are at least three "generations" of CAR compositions. In the first generation of CAR, the binding motif (e.g., single-chain fragment variable, binding motif) is connected or linked to the signaling domain (e.g., CD3ζ) via a transmembrane domain, optionally including a hinge domain and one or more spacers. In the second generation of CAR, a costimulatory domain (CM1, such as CD28, 4-1BB or OX-40) is introduced together with the signaling domain (e.g., CD3ζ). In the third generation of CAR, a second costimulatory domain (CM2) is included.

[0210] TCR is a heterodimer composed of α chain and β chain. TCR signaling requires the recruitment of signaling proteins that produce immune synapses. In addition, the positioning of TCR at the plasma membrane depends on the CD3 complex expressed in T cells. For example, using the transmembrane and signaling domains of the CAR construct, an engineered single-chain TCR can be generated, and the methods and constructs used for this are known (e.g., sTCR and TCR-CAR molecules, such as TCRβ chains and CD28TM and CD28 and CD3ζ fusion). The anti-CD20 and / or anti-CD19 antigen binding systems disclosed herein may include one or more antigen binding motifs for binding CD20 and / or CD19. In some embodiments, the antigen binding system further includes a costimulatory domain and / or an extracellular domain (e.g., a "hinge" or "interval" 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 the present disclosure comprises at least a binding motif that binds to human CD20, a costimulatory domain, an extracellular domain, a transmembrane domain, and a CD3-zeta or CD3-episilon activation domain.

[0211] In some embodiments, the antigen binding system of the present disclosure may comprise an antigen binding system comprising a leader peptide (P), a binding motif (B), an extracellular domain of a co-stimulatory protein (E), a transmembrane domain (T), a co-stimulatory domain (C), a second co-stimulatory domain (C') and an activation domain (A) One or more or all. In some cases, the antigen binding system is configured according to: BETA. In some cases, the antigen binding system is configured according to: PBETA. In some cases, the antigen binding system is configured according to: BETCA. In some cases, the antigen binding system is configured according to: PBETCA. In some cases, the antigen binding system is configured according to: BETC C'A. In some cases, the antigen binding system is configured according to: PBETC C'A. In some embodiments, the antigen binding system comprises VH and VL, optionally wherein the CAR is configured according to: P-VH-VL-ETCA or P-VL-VH-ETCA. In some embodiments, VH and VL are linked by a linker (L), optionally wherein the CAR is configured according to the following, from N-terminus to C-terminus: P-VH-L-VL-ETCA or P-VH-L-VL-ETCA.

[0212] One or more antigen binding motifs determine the target of the antigen binding system. The binding motif of the antigen binding system can include any binding motif, such as an antibody provided by the present disclosure, such as a binding motif of the present disclosure. In some embodiments, the binding motif can include an anti-CD20 binding motif and / or an anti-CD19 binding motif. In some embodiments, the binding motif can include an anti-CD20 binding motif and / or an anti-CD19 binding motif.

[0213] Binding motifs are used for chimeric antigen receptors at least in part because they can be engineered to be expressed as part of a single chain with other CAR components. See, for example, U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar 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, each document is incorporated herein by reference regarding the binding motif domains in CAR. Binding motifs or scFv are single-chain antigen binding fragments comprising heavy chain variable domains and light chain variable domains, which are connected or linked together. See, e.g., U.S. Pat. Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136, regarding binding motif domains, each of which is incorporated herein by reference. When derived from a parent antibody, the binding motif may retain some, all, or substantially retain the binding of the parent antibody to the target antigen.

[0214] The hinge can be an extracellular domain of an antigen binding system located between a binding motif and a transmembrane domain. A hinge can also be referred to as an extracellular domain or a "spacer". A hinge can contribute to receptor expression, activity and / or stability. In some embodiments, a hinge domain is located between a binding motif and a transmembrane domain. A hinge can also provide flexibility for accessing a targeted antigen. A hinge comprises an immunoglobulin-like hinge domain.

[0215] In some embodiments, the antigen binding system of the present disclosure may comprise a hinge that is, is from, or is derived from (e.g., comprises all or a fragment thereof) an immunoglobulin-like hinge domain. In some embodiments, the hinge domain is from or is derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinge or a fragment thereof of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM.

[0216] The hinge may be derived from a natural source or from a synthetic source. In some embodiments, the antigen binding system of the present disclosure may comprise a hinge that is, is from, or is derived from (e.g., comprises all or a fragment thereof) CD2, CD3 delta, CD3 epsilon, CD3gamma, CD4, CD7, CD8.alpha., CD8.beta., CD11a(ITGAL), CD11b(ITGAM), CD11c(ITGAX), CD11d(ITGAD), CD18(ITGB2), CD19(B4), CD27(TNFRS F7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48(SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM 1), 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), CD 158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD1 60(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), 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)、CD352(SLAMF6)、CD353(SLAMF8)、CD355(CRTAM)、CD357(TNFRSF18)、induced T cell co-stimulator(ICOS)、LFA-1(CD11a / CD18)、NKG2C、DAP-10、ICAM-1、NKp80(KLRF1)、IL-2R beta、IL-2R gamma、IL-7Ralpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor or Toll ligand receptor, or a fragment or combination thereof. In certain embodiments, CAR does not include a CD28 hinge.

[0217] In some embodiments, the antigen binding system of the present disclosure may include a hinge that is, is or is derived from (e.g., includes all or fragments thereof) a hinge of CD8 alpha. In some embodiments, the hinge is, is or is derived from a hinge of CD28. In some embodiments, the hinge is, is or is derived from a fragment of a hinge of CD8 alpha or a fragment of a hinge of CD28, wherein the fragment is anything less than the whole. In some embodiments, a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge comprises an amino acid sequence of a CD8 alpha hinge or a CD28 hinge that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or the C-terminus or both. Exemplary hinge sequences include those provided in Table 54 (SEQ ID NOs: 261-269).

[0218] The polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, the polynucleotide encoding the hinge domain comprises a polynucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the hinge domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.

[0219] In general, a "transmembrane domain" (e.g., of an antigen binding system) refers to a domain that has the property of being present in a membrane when present in a molecule at a cell surface or cell membrane (e.g., spanning a portion or all of a cell membrane). The co-stimulatory domain of an antigen binding system of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. It is not necessary for every amino acid in the transmembrane domain to be present in the membrane. For example, in some embodiments, the transmembrane domain is characterized in that a specified segment or portion of a protein is substantially located in the membrane. A variety of algorithms can be used to analyze amino acid or nucleic acid sequences to predict protein subcellular localization (e.g., transmembrane localization). Programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0220] The type of transmembrane domain included in the antigen binding system described herein is not limited to any type. In some embodiments, a transmembrane domain naturally associated with a binding motif and / or an intracellular domain is selected. In some cases, the transmembrane domain comprises one or more amino acid modifications (e.g., deletions, insertions and / or substitutions), such as to avoid such domains from being combined with the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0221] The transmembrane domain may be derived from a natural source or from a synthetic source. When the source is a natural source, the domain may be derived from any membrane-bound protein or transmembrane protein. Exemplary transmembrane domains may be derived from (e.g., may include at least one transmembrane domain thereof) the alpha, beta or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, 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-2Rbeta, IL-2R gamma, IL-7R alpha, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, 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 1molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (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, truncations or combinations thereof. In some embodiments, the transmembrane domain can be synthetic (and can, for example, primarily comprise hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan and valine is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly connected or linked to the cytoplasmic domain. In some embodiments, a short oligopeptide or polypeptide linker (e.g., between 2 and 10 amino acids in length) can form a connection between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet. ;

[0222] The polynucleotides and polypeptide sequences of the transmembrane domains provided herein are known. In some embodiments, the polynucleotides encoding the transmembrane domains comprise a polynucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the transmembrane domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100% or 95-100%) identical to a known polypeptide sequence. Optionally, a short spacer can form a connection between any or some extracellular, transmembrane and intracellular domains of CAR.

[0223] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains, which cause and / or mediate intracellular signals after the target antigen binds to the binding motif, for example, it activates one or more immune cell effector functions (e.g., natural immune cell effector functions). In some embodiments, the signaling 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 can be a cytolytic activity or auxiliary activity comprising cytokine secretion. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and / or other T cell functions. The intracellular domain may include a signaling domain as an activation domain. The intracellular domain may include a signaling domain as a co-stimulatory signaling domain.

[0224] Intracellular signaling domains that can transduce signals when an antigen binds to an immune cell are known, any of which can be included in the antigen binding system of the present disclosure. 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, e.g., Brownlie et al., Nature Rev. Immunol. 13: 257-269 (2013)).

[0225] In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcRgamma, FcR beta, CD3 zeta, CD3 gamma, CD3delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2: a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27: 591-619 (2009)).

[0226] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptors, immunoglobulin proteins, 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, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptors, 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-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, 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 death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocytic activation molecules (SLAM proteins), 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.

[0227] CAR can include a co-stimulatory signaling domain, for example, to increase signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), 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). The signal generated by TCR alone may not be sufficient to fully activate T cells, and secondary or co-stimulatory signals can increase activation. Therefore, in some embodiments, the signaling domain further includes one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., natural immune cell effector functions described herein). In some embodiments, a portion of such a co-stimulatory signaling domain can be used, as long as the portion transduces an effector function signal. 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 bound to CD83. Exemplary co-stimulatory proteins have amino acid sequences of co-stimulatory proteins naturally present on T cells, and the complete native amino acid sequences of the co-stimulatory proteins are described in NCBI reference sequence: NP_006130.1. In some cases, CAR includes a 41BB co-stimulatory domain encoded by a sequence according to SEQ ID NO: 270, as shown below:

[0228] SEQ ID NO:270

[0229] AGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGG ACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat

[0230] The polynucleotide and polypeptide sequences of the signaling domains provided herein are known. In some embodiments, the polynucleotide encoding the signaling domain comprises a polynucleotide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the signaling domain comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to a known polypeptide sequence.

[0231] In various embodiments, it is necessary to regulate (e.g., reduce) the mechanism of antigen binding system activity, for example to minimize or reduce adverse events caused by antigen binding system activity. It may also be necessary to include an inductive "on" or "accelerator" switch in immune cells. Suitable techniques include using inducible caspase-9 (U.S. application 2011 / 0286980) or thymidine kinase before, after, or simultaneously with transduction of cells with the CAR construct disclosed herein. Other methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other technologies.

[0232] According to the present disclosure, on-off or other types of control switch technologies can be incorporated herein. These technologies can include the use of dimerization domains and optional activators of such domain dimerization, such as Wu et al., Science 2014 350 (6258), which utilizes the FKBP / Rapalog dimerization system in certain cells, and the contents of which are incorporated herein by reference in their entirety. Additional dimerization technologies are described in, for example, 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 also incorporated herein by reference in terms of the contents of dimerization technologies. Additional dimerization pairs can include cyclosporine-A / cyclophilin receptors, estrogen / estrogen receptors (optionally using tamoxifen, 4-hydroxytamoxifen or endoxifen), glucocorticoids / glucocorticoid receptors, tetracycline / tetracycline receptors, and / or vitamin D / vitamin D receptors. Other examples of dimerization technology can be found in, for example, WO 2014 / 127261, WO 2015 / 090229, US2014 / 0286987, US2015 / 0266973, US2016 / 0046700, U.S. Patent No. 8,486,693, US2014 / 0171649 and US2012 / 0130076, the contents of which are further incorporated herein by reference in their entirety.

[0233] In some embodiments, the antigen binding systems of the present disclosure comprise a leader peptide (also referred to herein as a "signal peptide" or "leader sequence"). In certain embodiments, the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical to the amino acid sequence MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 249), MALPVTALLLPLALLLHAARP (SEQ ID NO: 250), or MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 295).

[0234] CAR components can be exchanged or "swapped" for equivalent components using conventional biotechnology techniques. Only to provide some non-limiting and partial examples, CAR of the present disclosure may include a combination of a binding motif as provided herein with a hinge as provided herein and a co-stimulatory domain as provided herein. In certain instances, CAR of the present disclosure may include a leader sequence as provided herein together with a binding motif as provided herein with a hinge as provided herein and a co-stimulatory domain as provided herein. In various embodiments, the present disclosure provides a combination of a binding motif according to any one of SEQ ID NOs: 251-260 and a hinge according to any one of SEQ ID NOs: 261-269 (eg, adjacent to it), optionally with SEQ ID NO: 270 41BB co-stimulatory domains further combined (eg, adjacent to it). Some non-limiting examples thereof are provided in SEQ ID NOs: 271-290.

[0235] Bicistronic CAR can include a first CAR sequence and a second CAR sequence expressed as a single polypeptide, and the single polypeptide includes a cleavable joint between the first and second CARs. An exemplary cleavable joint is furin-GSG-T2A (see, for example, Chng et al. MAbs. 2015 Mar-Apr; 7 (2): 403-412, which is incorporated herein by reference for aspects of cleavable joints; see also Guedan et al. Mol Ther Methods Clin Dev. 2019 Mar 15; 12: 145-156, which is incorporated herein by reference for aspects of bicistronic CAR design). To provide only one non-limiting example of a bicistronic CAR structure, a bicistronic CAR may comprise (a) a first CAR comprising (i) a signal peptide (e.g., a 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 or hinge (e.g., a CD28T spacer); (vi) a transmembrane domain (e.g., a CD28 transmembrane domain); (vii) a costimulatory domain (e.g., a CD28 costimulatory domain); (viii) a stimulatory domain (e.g., a CD3z stimulatory domain); (b) a cleavable linker (e.g., a furin GSG-T2A linker); and (c) a second CAR comprising (i) a signal peptide (e.g., 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 SEQ ID NO: 307-313 of any one of the linker); (iv) the anti-CD20 light chain variable domain of the present disclosure; (v) spacer or hinge (e.g., CD8a spacer); (vi) transmembrane domain (e.g., CD8 transmembrane domain); (vii) costimulatory domain (e.g., 41bb costimulatory domain); and (viii) stimulatory domain (e.g., CD3z stimulatory domain). Therefore, but not limited to, exemplary anti-CD20 / anti-CD19 bicistronic CARs may have or be included in the nucleotide and amino acid sequences shown in SEQ ID NO: 291 and 292.

[0236] Bispecific CAR can be a single polypeptide comprising the first binding motif of the present disclosure and the second binding motif as an anti-CD19 binding motif. In a non-limiting exemplary embodiment, bispecific CAR can include (i) leader (e.g., CSF2RA signal peptide), (ii) anti-CD20 light chain variable domain of the present disclosure;(iii) joint;(iv) anti-CD20 heavy chain variable domain;(v) joint (e.g., truncated joint);(vi) anti-CD19 light chain variable domain;(vii) joint;(viii) anti-CD19 heavy chain variable domain;(ix) extracellular domain (e.g., CD28T hinge or IgG4 hinge);(x) transmembrane domain (e.g., CD28 transmembrane domain);(xi) intracellular region (e.g., CD28 intracellular costimulatory domain and / or 41bb costimulatory domain); and stimulatory domain (e.g., CD3z stimulatory domain). Thus, but not limited to, an exemplary anti-CD20 / anti-CD19 bispecific CAR may have or comprise the nucleotide and amino acid sequences shown in SEQ ID NOs: 293-306.

[0237] Various CAR sequences, components and / or frameworks are known, including but not limited to sequences of hinges, spacers, transmembrane domains, co-stimulatory domains, stimulatory domains, binding motifs and their respective variants, and if, for example, heavy chain variable domain sequences or CDR sequences and light chain variable domain sequences or CDR sequences are provided, a CAR with the desired binding and components or frameworks can be easily constructed.

[0238] The present 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 an anti-CD20 binding motif of the present disclosure and an anti-CD19 binding motif of the present disclosure. In some embodiments, the bispecific antibody comprises an anti-CD20 binding motif comprising an anti-CD20 heavy chain variable domain of the present disclosure and an anti-CD20 light chain variable domain of the present disclosure, and an anti-CD19 binding motif comprising an anti-CD19 heavy chain variable domain and an anti-CD19 light chain variable domain.

[0239] The present disclosure includes conjugates in which the antibodies of the present disclosure are associated with a therapeutic agent or a detectable moiety. In various embodiments, the therapeutic agent is an anticancer agent provided herein. In certain embodiments, the conjugates provided include one or more detectable moieties, i.e., are "labeled" with one or more such moieties. In some such embodiments, the conjugates of the present 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 for labeled antibody conjugates described herein. Suitable detectable moieties include, but are not limited to, various ligands, radionuclides; fluorescent dyes; chemiluminescent agents (such as, for example, acridinium esters, stable dioxetanes, etc.); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (such as gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (such as, for example, dyes, colloidal gold, etc.); biotin; digoxigenin; haptens; and proteins for which antisera or monoclonal antibodies can be obtained.

[0240] The present disclosure includes nucleic acids encoding anti-CD20 binding motifs and / or anti-CD19 binding motifs provided herein. The present 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). The present 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 that bind CD20 and CD19). The nucleic acid sequence of SEQ ID NO:2 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NOs:1 and 3-11. The nucleic acid sequence of SEQ ID NO:13 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NOs:12 and 14-22. The nucleic acid sequence of SEQ ID NO:24 includes and provides an exemplary nucleic acid sequence corresponding to and encoding each of SEQ ID NOs: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 NOs: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 NOs: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 NOs: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 NOs: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 NOs: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 NOs: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 NOs: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 NOs: 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 NOs: 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 NOs: 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 NOs: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 NOs: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 NOs: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 NOs: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 NOs: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 NOs: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 NOs:210 and 212-220. The present disclosure includes nucleic acids encoding the anti-CD19 binding motifs 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 NOs: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 NOs:232 and 234-242.

[0241] The present disclosure includes a vector comprising a nucleic acid of the present disclosure and / or a polypeptide encoding the present disclosure. In various embodiments, the present disclosure includes a vector comprising a nucleic acid encoding an anti-CD20 binding motif and / or an anti-CD19 binding motif provided herein. In various embodiments, the present disclosure includes a vector comprising a nucleic acid encoding an antibody provided herein, including but not limited to a nucleic acid encoding a binding motif molecule (e.g., an anti-CD20 binding motif or an anti-CD19 binding motif). In various embodiments, the present disclosure includes a vector comprising a nucleic acid encoding one or more antigen binding systems provided herein, including but not limited to a nucleic acid encoding a bicistronic or bispecific chimeric antigen receptor (e.g., a bicistronic and bispecific chimeric antigen receptor binding CD20 and CD19).

[0242] Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo 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.

[0243] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for breeding and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, 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 the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, a bicistronic IRES vector (eg, from Clontech) is used to contain nucleic acids encoding an antigen binding system and an inducible expression construct described herein.

[0244] In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alphavirus, vaccine, adenovirus, adeno-associated virus, herpes virus and fowl pox virus vectors, and preferably have the ability to transform immune cells (e.g., T cells) naturally or engineered.

[0245] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to include a replication system that functions in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0246] The recombinant expression vector may comprise one or more marker genes that allow selection of transformed or transfected hosts. The marker gene comprises antimicrobial resistance, such as resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, etc. Suitable marker genes of the recombinant expression vector comprise, for example, neomycin / G418 resistance genes, puromycin resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0247] Useful vectors in the context of the present disclosure may be "naked" nucleic acid vectors (i.e., vectors with little or no protein, sugar, and / or lipids encapsulating them), or vectors complexed with other molecules. Other molecules that may be suitably combined with vectors include, but are not limited to, viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties, such as ligands, receptors, or antibodies that target cellular molecules.

[0248] 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 guns, nanoparticle-mediated delivery or electroporation. Transduction includes viral delivery of vectors to cells, such as by vectors disclosed herein, including but not limited to retroviruses, lentiviruses and AAV.

[0249] The present 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 the present disclosure. In some embodiments, the method includes transducing a cell with a vector comprising a polynucleotide encoding at least one antigen binding system. The present disclosure includes cells that contain or are transformed to contain at least one vector encoding one or more polypeptides of the present disclosure. The present 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. The present disclosure includes cells that contain or are transformed to contain at least one vector encoding an antibody provided herein, the antibody including but not limited to a binding motif molecule (e.g., an anti-CD20 binding motif or an anti-CD19 binding motif). The present disclosure includes cells that contain or are transformed to contain at least one vector encoding one or more antigen binding systems provided herein, the antigen binding system including but not limited to a bicistronic or bispecific chimeric antigen receptor (e.g., a bicistronic or bispecific chimeric antigen receptor that binds CD20 and CD19). In some embodiments, cells are co-transfected or co-transduced with two vectors, each vector encoding a different CAR, and the two different CARs are bicistronic CARs together. Cells transfected or transduced with two different vectors (which encode two different CARs as bicistronic CARs together) can be performed simultaneously on a single cell population, can be performed simultaneously on two different cell populations, wherein each cell population is transduced with only one of the two vectors, or can be performed independently on two different cell populations, each cell population is transduced with only one of the two vectors.

[0250] The present disclosure includes cells comprising one or more polypeptides of the present disclosure. The present disclosure includes cells comprising (e.g., expressing) anti-CD20 binding motifs and / or anti-CD19 binding motifs provided herein. The present disclosure includes cells comprising (e.g., expressing) antibodies provided herein, including but not limited to binding motifs (e.g., anti-CD20 binding motifs or anti-CD19 binding motifs). The present disclosure includes cells comprising (e.g., expressing) one or more antigen binding systems provided herein, including but not limited to bicistronic or bispecific chimeric antigen receptors (e.g., bicistronic and bispecific chimeric antigen receptors that bind CD20 and CD19).

[0251] In other aspects, cells comprising polynucleotides or vectors disclosed herein are provided herein. In some embodiments, the disclosure relates to cells, such as in vitro cells, comprising polynucleotides encoding CARs or TCRs comprising one or both of the scFv disclosed herein. In other embodiments, the disclosure relates to cells, such as in vitro cells, comprising polypeptides encoded by CARs or TCRs comprising one or both of the scFv disclosed herein. In some embodiments, the polypeptide comprises the following amino acid sequence or any combination thereof.

[0252] SEQ ID NO: 232 (anti-CD19 scFv light chain):

[0253] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0254] SEQ ID NO: 221 (anti-CD19 scFv heavy chain):

[0255] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0256] SEQ ID NO:56 (anti-CD20 light chain):

[0257] DIQMTQSPSSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

[0258] SEQ ID NO:45 (anti-CD20 heavy chain):

[0259] QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSS

[0260] SEQ ID NO:56 (anti-CD20 scFv 2 light chain):

[0261] DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

[0262] SEQ ID NO:155 (Anti-CD20 scFv 2 heavy chain):

[0263] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSRYVWSWIRQPPGKGLEWIGEIDSSGKTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

[0264] SEQ ID NO:144 (Anti-CD20 scFv 3 light chain):

[0265] DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK

[0266] SEQ ID NO:177 (Anti-CD20 scFv 3 heavy chain):

[0267] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYAWSWIRQPPGKGLEWIGEIDHRGFTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

[0268] SEQ ID NO:78 (Anti-CD20 scFv 4 light chain):

[0269] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK

[0270] SEQ ID NO:67 (Anti-CD20 scFv 4 heavy chain):

[0271] QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS

[0272] For the polynucleotides, vectors or polypeptides disclosed herein, any cell can be used as a host cell. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, true bacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, such as Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia discolor. marcescans and Shigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonas such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of: T cells, B cells, tumor infiltrating lymphocytes (TIL), 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 cell is a T cell. In another embodiment, the immune cell is a NK cell. In certain embodiments, the T cell is a tumor infiltrating lymphocyte (TIL), an autologous T cell, an engineered autologous T cell (eACT TM ), allogeneic T cells, heterologous T cells, or any combination thereof.

[0273] Chimeric antigen receptors (CAR or CAR-T) and engineered T cell receptors (TCR) can be easily inserted into immune cells (eg, T cells) and expressed by them to produce binding agents. In certain embodiments, cells (eg, immune cells such as T cells) are obtained from donor subjects. In some embodiments, donor subjects are human patients with cancer or tumors. In other embodiments, donor subjects are human patients who do not suffer from cancer or tumors. In some embodiments, engineered cells are autologous to the subject. In some embodiments, engineered cells are allogeneic to the subject.

[0274] The cells of the present disclosure may be obtained from any source known in the art. For example, T cells may be differentiated from a hematopoietic stem cell population in vitro, or T cells may be obtained from a subject. T cells may be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained using any number of techniques known to those skilled in the art (e.g., FICOLL TM The cells are collected from a blood unit collected from a subject by separation and / or apheresis. In certain embodiments, the cells collected by apheresis are washed to remove the plasma portion and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, the centrifuge can be separated, for example, by using a semi-automatic non-reverse flow centrifuge (e.g., Cobe TM 2991 Cell Processor, BaxterCytoMate TM In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other saline solutions with or without buffers. In certain embodiments, undesired components of the aliquot sample are removed. Additional 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.

[0275] In certain embodiments, the erythrocytes are lysed and the monocytes are depleted (e.g., via PERCOLL TM Gradients, separation by centrifugation) are used to separate T cells from PBMCs. In some embodiments, specific subsets of T cells, such as CD4 + 、CD8 + 、CD28 + 、CD45RA + and CD45RO +T cells can be further isolated by 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 against surface markers specific to the negatively selected cells. In some embodiments, cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry (which uses a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells) can be used. For example, to enrich CD4 + Cells, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20 and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

[0276] In some embodiments, PBMC is directly used for the genetic modification (such as CAR or TCR) of immune cells using methods as described herein. In certain embodiments, after separating PBMC, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes are sorted into initial T cells, memory T cells and effector T cell subsets before or after genetic modification and / or amplification.

[0277] In some embodiments, by identifying + CD8 + The cells are further sorted into naive cells, central memory cells, and effector cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and is granzyme B negative. In some embodiments, central memory T cells are CD8 + 、CD45RO + and CD62L + In some embodiments, the effector T cells are negative for CCR7, CD28, CD62L, and CD127 and positive for granzyme B and perforin. In certain embodiments, the CD4 + T cells can be further sorted into subsets. For example, CD4 + T helper cells are sorted into naive cells, central memory cells, and effector cells.

[0278] In some embodiments, immune cells such as T cells are genetically modified using known methods after separation, or immune cells are activated and amplified in vitro (or, differentiated in the case of progenitor cells) before being genetically modified. 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 comprising one or more nucleotide sequences encoding CAR), and then activated and / or amplified in vitro. Methods for activating and amplifying T cells are known in the art and described in, for example, 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 as a whole. In general, such methods include contacting PBMCs or separated T cells with stimulants and co-stimulators (e.g., anti-CD3 and anti-CD28 antibodies, typically adhered to beads or other surfaces) in a culture medium with appropriate cytokines (e.g., IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same beads act as "surrogate" antigen presenting cells (APCs). An example is System, CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, the methods described in, for example, U.S. Pat. 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 activate and stimulate T cells to proliferate with feeder cells and appropriate antibodies and cytokines.

[0279] In various aspects of the present disclosure, cells that contain, express, encode or are transformed to encode a vector or polypeptide of the present disclosure (e.g., an anti-CD20 binding motif and / or an anti-CD20 / anti-CD19 antigen binding system of the present disclosure) are binding agents. A binding agent or a population of binding agents can be used as (e.g., as an active agent thereof) a binding agent (compositions comprising cells that can be used as a treatment, e.g., for cancer).

[0280] The present disclosure further includes methods and processes for producing antibody agents as disclosed herein, for example, by transforming (e.g., transducing) cells with vectors or nucleic acids disclosed herein. In some embodiments, the method or process for producing antibody agents as disclosed herein comprises transforming (e.g., transducing) cells with nucleic acids encoding at least one antigen binding system provided herein (e.g., nucleic acids present in vectors). In general, the antibody agents described herein can be produced by immune cells, such as cells that can be used or can be used for adoptive cell therapy. In some embodiments, the binding agent is produced by a cell type selected from the group consisting of: TIL, T-cells, CD8 + cells, CD4 +T cells, NK cells, gamma-delta T cells, regulatory T cells, or peripheral blood mononuclear cells. "Tumor infiltrating lymphocytes" or TILs are white blood cells that have left the bloodstream and migrated into the tumor. Lymphocytes can be divided into three groups, which include B cells, T cells, and natural killer cells. "T cells" are CD3 + cells, which contain CD4 + Helper cells, CD8 + Cytotoxic T cells and gamma-delta T cells.

[0281] In certain embodiments, the binding agent is produced by genetically modifying (e.g., transforming) cells, such as immune cells, with nucleic acids encoding an antigen binding system and / or expression constructs described herein (e.g., (i) a first recombinant expression vector comprising a nucleic acid encoding an antigen binding system and a second recombinant expression vector comprising an inducible expression construct, (ii) a single recombinant expression vector comprising both a nucleic acid encoding an antigen binding system and an inducible expression construct; or (iii) a recombinant expression vector comprising a constitutive expression construct). Recombinant expression vectors can contain any type of nucleotides, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and can contain natural, non-natural, or altered nucleotides. Recombinant expression vectors can contain naturally occurring or non-natural internucleotide linkages, or both types of linkages.

[0282] In some embodiments, the method comprises transducing a cell with a polynucleotide encoding an antigen binding system as disclosed herein. In some embodiments, the method comprises transducing a cell with a vector comprising a polynucleotide encoding an antigen binding system.

[0283] In some embodiments, donor T cells for T cell therapy are obtained from patients (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from non-patient subjects. In exemplary aspects, subjects can undergo leukapheresis, wherein ex vivo collection, enrichment or depletion of leukocytes to select and / or separate cells of interest, such as T cells. These T cell isolates can be amplified and processed by methods, so that one or more CAR constructs of the present disclosure can be introduced, thereby producing CAR T cells of the present disclosure.

[0284] In some embodiments, immune cells are obtained from a subject and transformed, e.g., transduced, with an inducible expression construct or constitutive expression construct as described herein, e.g., an expression vector comprising an inducible expression construct or constitutive expression construct as described herein, to obtain a binding agent. Thus, in some embodiments, the binding agent comprises autologous cells administered to the same subject from which the immune cells were obtained. In some embodiments, immune cells are obtained from a subject and transformed, e.g., transduced, with an inducible expression construct or constitutive expression construct as described herein, e.g., an expression vector comprising an inducible expression construct or constitutive expression construct as described herein, to obtain a binding agent that is allogeneically transferred to another subject.

[0285] In certain embodiments, T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In other embodiments, the donor subject is a human patient who does not suffer from cancer or a tumor.

[0286] Various compositions disclosed herein include engineered cell populations, which can be produced by any means. In some embodiments, the disclosure provides a human cell population engineered to express an antigen binding system as described herein. In some embodiments, such a population includes a binding agent. In some embodiments, such a population includes a cultivated population. In some embodiments, such a population is a cell culture population from a single human source, and the single human source can accept the administration of a culture population in some embodiments. As disclosed herein, a binding agent can include any single cell or cell population, for example, an engineered cell population as provided herein.

[0287] Other aspects of the present disclosure relate to compositions comprising polynucleotides as described herein, vectors as described herein, polypeptides as described herein, or in vitro cells as described herein. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, a diluent, a solubilizer, an emulsifier, a preservative, and / or an 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 as described herein. In another embodiment, the composition comprises a CAR or TCR containing a TCD encoded by a polynucleotide of the present disclosure. In another embodiment, the composition comprises a T cell containing a CAR or TCR (which comprises one or two scFv disclosed herein).

[0288] In other embodiments, the composition is selected for parenteral delivery, for inhalation or for delivery through the digestive tract, such as oral. It is within the ability of those skilled in the art to prepare such pharmaceutically acceptable compositions. In certain embodiments, buffer is used to maintain the composition at physiological pH or slightly lower pH, generally in the pH range of about 5 to about 8. In certain embodiments, when considering parenteral administration, the composition is a pyrogen-free, parenterally acceptable aqueous solution form, which is included in a pharmaceutically acceptable vehicle with or without the composition described herein of another therapeutic agent. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, wherein the composition described herein (with or without at least one other therapeutic agent) is formulated into a sterile isotonic solution properly preserved. In certain embodiments, preparation involves a preparation of a desired molecule of a polymeric compound (such as polylactic acid or polyglycolic acid), a pearl or a liposome providing a controlled or sustained release of the product, and then the preparation is delivered via a storage injection (depot injection). In certain embodiments, an implantable drug delivery device is used to introduce the desired molecule.

[0289] In some embodiments, the disclosure provides pharmaceutical compositions that contain and / or deliver one or more of the disclosure, e.g., the antigen binding systems of the disclosure, nucleic acids encoding them, and / or cells or populations thereof that contain and / or express them.

[0290] In some embodiments, the disclosure provides a pharmaceutical composition 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 CAR (i.e., "binding agent pharmaceutical composition"). The binding agent pharmaceutical composition may include one or more cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include 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.

[0291] The binding agent pharmaceutical composition of the present disclosure can be formulated for administration according to any embodiment set forth herein, at least one non-limiting example of which is intravenous administration. The composition can be formulated for intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural and / or subcutaneous administration routes. Preferably, the composition is formulated for parenteral administration routes. Compositions suitable for parenteral administration can be aqueous or non-aqueous isotonic sterile injections, which can contain antioxidants, buffers, antibacterial agents and solutes, such as making the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions can contain one or more suspending agents, solubilizers, thickeners, stabilizers and preservatives. The binding agent pharmaceutical composition of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented).

[0292] The sterile composition for injection can be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. 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 with a suitable solubilizer, such as alcohols such as ethanol, and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 TM , HCO-50 and other combinations.

[0293] Non-limiting examples of oily liquids include sesame oil and soybean oil, and it can be combined with benzyl benzoate or benzyl alcohol to become a solubilizing agent. Other items that can 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 prepared injection can be packaged in a suitable ampoule.

[0294] In one embodiment, the binding agent pharmaceutical composition is substantially free of detectable levels of contaminants, such as endotoxins, mycoplasmas, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria and fungi. In one embodiment, the bacteria is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and / or Group A Streptococcus pyogenes.

[0295] In various embodiments, cells provided herein (e.g., binding agents, such as engineered T cells or engineered NK cells) can be activated and / or amplified from binding agent pharmaceutical compositions, and / or produce binding agent pharmaceutical compositions. In some embodiments, additional steps can be performed before being applied to a subject. For example, before being applied to a subject, after immunocytes are contacted (e.g., transduced or transfected) with inducible expression constructs or constitutive expression constructs as described herein (e.g., expression vectors comprising inducible expression constructs or constitutive expression constructs), binding agents can be amplified in vitro. Before being applied to a subject, in vitro amplification can be performed for 1 day or more, such as 2 days or more, 3 days or more, 4 days or more, 6 days or more, or 8 days or more. In some embodiments, in vitro amplification can be performed for 21 days or less, such as 18 days or less, 16 days or less, 14 days or less, 10 days or less, 7 days or less, or 5 days or less. For example, in vitro amplification can be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days before being applied to a subject. A binding agent pharmaceutical composition comprising, for example, a binding agent (e.g., an engineered T cell or an engineered NK cell) can be formulated for administration at a desired dose, e.g., 10 4 Up to 10 9 cells / kg body weight (e.g. 10 5 Up to 10 6 Certain embodiments of the present disclosure include methods of administering to a subject a pharmaceutical composition as described herein, such as, for example, a binding agent as described herein (e.g., an engineered cell population of the present disclosure), a protein therapeutic as described herein, a composition comprising a binding agent, and / or a composition comprising a protein therapeutic, e.g., in an amount effective to treat the subject when administered with an appropriate dosing regimen.

[0296] In some embodiments, the binding agent is autologous to the subject, and before the immune cells are isolated from the subject, the subject can be immunologically naive, immune, diseased or in another condition. In some embodiments, during in vitro expansion, the binding agent can be stimulated with an antigen (e.g., a TCR antigen). Antigen-stimulated amplification can optionally be supplemented with amplification under conditions that nonspecifically stimulate lymphocyte proliferation, such as, for example, anti-CD3 antibodies, anti-Tac antibodies, anti-CD28 antibodies or phytohemagglutinin (PHA). The amplified binding agent can be directly administered to the subject or can be frozen for future use, i.e., for subsequent administration to the subject.

[0297] In some embodiments, the binding agent is treated with interleukin-2 (IL-2) ex vivo prior to infusion into the cancer patient, and the cancer patient is treated with IL-2 after infusion. In addition, in some embodiments, the cancer patient may undergo preliminary lymphodepletion-temporary ablation of the immune system prior to administration of the binding agent. The combination of IL-2 treatment and preliminary lymphodepletion can enhance the persistence of the binding agent. In some embodiments, the binding agent is transduced or transfected with a nucleic acid encoding a cytokine, which can be engineered to provide constitutive, regulatable, or time-controlled expression of the cytokine. Suitable cytokines include, for example, cytokines that act to enhance the survival of T lymphocytes during the contraction phase, which can promote the formation and survival of memory T lymphocytes.

[0298] In certain embodiments, the binding agent is administered prior to, substantially simultaneously with, or after administration of another therapeutic agent, such as a cancer therapeutic agent. The cancer therapeutic agent can be, for example, a chemotherapeutic agent, a biological agent, or radiation therapy. In some embodiments, the subject receiving the binding agent is not administered a treatment sufficient to cause immune cell depletion, such as lymphodepleting chemotherapy or radiation therapy.

[0299] In some embodiments, the dosage administered to a subject may vary with the embodiment, the composition employed, the method of administration, and the site and subject being treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the therapeutically effective amount of the composition to be administered to a person or other subject to treat or prevent a medical condition. The precise amount of the composition required for therapeutic effectiveness may depend on a variety of factors, such as, for example, the activity of the binding agent and the route of administration.

[0300] A suitable number of binding agent cells may be administered to a subject. Although a single binding agent cell as described herein is capable of expanding and providing therapeutic benefit, in some embodiments, administration of 10 2 or more, e.g. 10 3 or more, 10 4 or more, 10 5 or more, or 10 8 In some embodiments, 10 12 or less, e.g. 10 11 or less, 10 9 or less, 10 7 or less, or 10 5 or fewer of the binding agent cells described herein. In some embodiments, administration of 10 2 -10 5 , 10 4 -10 7 , 10 3-10 9 or 10 5 -10 10 binder cells. For example, 10 4 Up to 10 9 cells / kg body weight (e.g. 10 5 Up to 10 6 The dosage of the binding agent pharmaceutical composition can be about 2×10 cells / kg body weight. 6 cells / kg, about 3X10 6 cells / kg, about 4X10 6 cells / kg, about 5X10 6 cells / kg, about 6X10 6 cells / kg, about 7X10 6 cells / kg, about 8X10 6 cells / kg, about 9X10 6 cells / kg, about 1X10 7 cells / kg, about 2X10 7 cells / kg, about 3X10 7 cells / kg, about 4X10 7 cells / kg, about 5X10 7 cells / kg, about 6X10 7 cells / kg, about 7X10 7 cells / kg, about 8X10 7 cells / kg or about 9X10 7 The binding agent pharmaceutical composition is administered at a dose of 1 cell / kg.

[0301] The dosage of the binding agent described herein can be administered to a mammal at once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, biweekly, semi-monthly, bimonthly, semi-monthly or annually as needed. The dosage unit containing an effective amount of the binding agent can be administered in a single daily dose, or the total daily dose can be administered in two, three, four or more divided doses administered daily as needed.

[0302] The practitioner can select a suitable mode of administration. The route of administration can be parenteral, for example, by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the composition is selected for parenteral delivery, inhalation or delivery through the digestive tract, such as oral administration. The dosage and method of administration can vary according to the weight, age, condition, etc. of the subject and can be appropriately selected.

[0303] In various embodiments, the binding agents described herein can be incorporated into pharmaceutical compositions. Pharmaceutical compositions comprising the binding agents of the present disclosure can 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 binding agents of the present disclosure can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Compositions comprising the binding agents of the present disclosure may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0304] In various embodiments, compositions containing the binding agents described herein, such as sterile preparations for injection, can be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. 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 with suitable solubilizers, such as alcohols such as ethanol, and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 TM , HCO-50, etc. As disclosed herein, the pharmaceutical composition comprising the binding agent can be in any form. Such forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[0305] The selection or use of any form may depend in part on the intended mode of administration and therapeutic application. For example, a composition comprising a binding agent of the present invention intended for systemic or local delivery may be in the form of an injectable or infusible solution. Therefore, a composition comprising a binding agent of the present invention may be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal or intramuscular injection). Parenteral administration refers to administration methods other than enteral and topical administration, typically by injection, and includes but is not limited to intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracervical and intrasternal injection and infusion.

[0306] The administration route may be parenteral, such as by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration may be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.

[0307] In various embodiments, the pharmaceutical composition comprising the binding agent of the present invention can be formulated into a solution, a microemulsion, a dispersant, a liposome or other ordered structure suitable for stable storage at high concentrations. A sterile injectable solution can be prepared by incorporating a composition comprising the binding agent of the present invention in a desired amount into a suitable solvent together with a combination of one or more components listed above, followed by filtration sterilization as required. Typically, a dispersant is prepared by incorporating a composition comprising the binding agent of the present invention into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, the preparation method includes vacuum drying and freeze drying of a powder producing a composition, the powder of the composition comprising the binding agent of the present invention plus any additional required components from its previously sterile filtered solution (see below). The appropriate fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersant, and by using a surfactant. Prolonged absorption of injectable compositions containing a binding agent of the present disclosure can be brought about by including in the composition containing a binding agent of the present disclosure an agent that delays absorption, for example, monostearate salts and gelatin.

[0308] The pharmaceutical composition comprising the binding agent of the present invention 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, the pharmaceutical composition comprising the binding agent of the present invention can be prepared by appropriately combining the therapeutic molecule with a pharmaceutically acceptable vehicle or medium, such as sterile water and saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, adhesive, and then mixing in the unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is the 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 to form a solubilizer. Other items that can 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 prepared injection can be packaged in a suitable ampoule.

[0309] In some embodiments, compositions comprising binding agents of the present disclosure can be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In some embodiments, compositions comprising binding agents of the present 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, 10 months, 11 months, 1 year, 11 / 2 years, or 2 years). Thus, in some embodiments, compositions comprising binding agents of the present disclosure are stably stored at 2-8°C (e.g., 4°C) for at least 1 year.

[0310] In some cases, pharmaceutical compositions comprising the binding agents of the present disclosure can be formulated into solutions. In some embodiments, compositions comprising the binding agents of the present disclosure can be formulated into buffered solutions, for example, at suitable concentrations and suitable for storage at 2-8°C (e.g., 4°C). Pharmaceutical compositions comprising binding agents as described herein can be formulated in immunoliposome compositions. Liposomes with extended circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0311] In certain embodiments, compositions comprising the binding agents of the present disclosure can be formulated with carriers that protect the compositions from rapid release, such as controlled release formulations comprising implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are known. See, for example, JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0312] In some embodiments, the composition comprising the binding agent of the present disclosure can be formulated into a composition suitable for intrapulmonary administration (e.g., administration via an inhaler or nebulizer) to a mammal such as a human. Methods for formulating such compositions are known. Dry powder inhaler formulations and suitable systems for formulation administration are also known. Pulmonary administration can be oral and / or nasal administration. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, a composition comprising a binding agent of the present disclosure can be administered to the lungs of a subject via a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are known and include, but are not limited to: (AstraZeneca; London, England) Inhaler( Cambridge, Mass.); (GlaxoSmithKline; London, England); and ECLIPSE TM (Sanofi-Aventis; Paris, France). See also, for example, PCT Publication Nos. WO 04 / 026380, WO 04 / 024156 and WO 01 / 78693. DPI devices have been used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, compositions comprising binding agents of the present disclosure can be administered intrapulmonary by a metered dose inhaler. These inhalers rely on a propellant to deliver discrete doses of the molecule to the lungs. Additional devices and methods of intrapulmonary administration are described, for example, in U.S. Patent Application Publication Nos. 20050271660 and 20090110679, each of which is incorporated herein by reference in its entirety.

[0313] In some embodiments, compositions comprising the binding agents of the present disclosure can be formulated for delivery to the eye, for example in the form of a pharmaceutically acceptable solution, suspension, or ointment. Preparations for treating the eye can be in the form of a sterile aqueous solution containing, for example, additional ingredients, such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, and viscosity increasing agents. The preparations described herein can be topically applied to the eye of a subject in need of treatment (e.g., a subject suffering from AMD) by conventional methods, for example, in the form of drops containing one or more compositions, or by bathing the eyes in a treatment solution.

[0314] In certain embodiments, a variety of devices for introducing drugs into the vitreous cavity of the eye may be suitable for administering compositions comprising the binding agents of the present disclosure. For example, U.S. Publication No. 2002 / 0026176 describes a plug containing a drug that can be inserted through the sclera so that it protrudes into the vitreous cavity to deliver the agent into the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantable device for introducing the suprachoroidal space or avascular region to continuously release the 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. Additional methods and devices for delivering therapeutic agents to the eye (e.g., transscleral patches and delivery via contact lenses) are described, for example, in Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181; Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10); Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347; Kim et al. (2007) Ophthalmic Res 39:244-254; and PCT Publication No. WO 04 / 073551, the disclosures of which are incorporated herein by reference in their entirety.

[0315] In various embodiments, subcutaneous administration can be accomplished by a device such as a syringe, a prefilled syringe, an automatic injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with a subcutaneous infusion set, or other devices for subcutaneous administration in combination with the binding agent drug.

[0316] The injection system of the present disclosure can adopt a delivery pen as described in U.S. Patent No. 5,308,341. Pen devices are generally used to deliver insulin to patients with diabetes. Such devices may include at least one injection needle (e.g., a 31-gauge needle of about 5 to 8 mm in length), which is generally pre-filled with one or more therapeutic unit doses of a therapeutic solution, and can be used to deliver the solution quickly to the subject with as little pain as possible. A drug delivery pen includes a vial holder, in which a vial of a therapeutic agent or other drug can be accommodated. The pen can be a completely mechanical device, or it can be combined with an electronic circuit to accurately set and / or indicate the dose of the drug injected into the user's body. See, for example, U.S. Patent No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the kit includes one or more disposable replacement needles. Pen devices suitable for delivering any one of the current characteristic compositions of the binding agent disclosed herein are also described in, for example, U.S. Patent Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of which are each incorporated herein by reference in their entirety. Microneedle-based pen devices are described, for example, in U.S. Pat. No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the precision pen injector (PPI) device MOLLY manufactured by Scandinavian Health Ltd. TM .

[0317] In some embodiments, a composition comprising a binding agent of the present disclosure may be delivered to a subject by a local administration that does not rely on the transport of the binding agent via the vascular system to its intended target tissue or site. For example, a composition comprising a binding agent of the present disclosure may be delivered by injection or implantation of a composition comprising a binding agent of the present disclosure or by injection or implantation of a device containing a composition comprising a binding agent of the present disclosure. In certain embodiments, after local administration near a target tissue or site, a composition comprising a binding agent of the present disclosure or one or more components thereof may diffuse to an intended target tissue or site that is not the site of administration.

[0318] In some embodiments, the composition comprising the binding agent of the present disclosure may be applied topically to a joint, e.g., directly to the joint (e.g., into the joint space) or near a joint. Examples of intra-articular joints to which the composition comprising the binding agent of the present disclosure may be topically applied include, e.g., hip, knee, elbow, wrist, sternoclavicular, temporomandibular, carpal, tarsal, ankle, and any other joint that suffers from an arthritic condition. The composition comprising the binding agent of the present disclosure may also be applied to a bursa, such as, e.g., acromion, biceps radialis, elbow radialis, deltoid, infrapatellar, ischium, and any other bursa.

[0319] In some embodiments, provided herein are compositions comprising a binding agent of the present disclosure in a unit dosage form that may be suitable for self-administration. Such unit dosage forms may be provided in containers, typically such as vials, cartridges, pre-filled syringes, or disposable pens. Dosage devices (such as the dosage device described in U.S. Patent No. 6,302,855) may also be used, for example, with injection systems as described herein.

[0320] The pharmaceutical solution may comprise a therapeutically effective amount of a composition comprising a binding agent of the present invention. Such an effective amount may be readily determined based in part on the effect of the composition comprising the binding agent of the present invention administered, or the combined effect of the composition comprising the binding agent of the present invention and one or more additional active agents (if more than one agent is used). The therapeutically effective amount of a composition comprising a binding agent of the present invention may also vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the composition (and one or more additional active ingredients) to elicit a desired response in the individual, such as improving at least one condition parameter, such as improving at least one symptom of a complement-mediated disorder. For example, a therapeutically effective amount of a composition comprising a binding agent of the present invention may inhibit (reduce its severity or eliminate its occurrence) and / or prevent any symptom of a disorder and / or disorder. A therapeutically effective amount is also a therapeutically effective amount in which the therapeutically beneficial effects exceed any toxic or deleterious effects of the composition comprising the binding agent of the present invention.

[0321] Compositions comprising a binding agent of the present disclosure can be used as a fixed dose or in mg / kg (mg / kg) dosage. In some embodiments, dosage can also be selected to reduce or avoid the generation of antibodies or other host immune responses for one or more antigen binding molecules in the composition comprising a binding agent of the present disclosure. Although it is never intended to limit, the exemplary dosage of a binding agent such as a composition comprising a binding agent of the present disclosure includes, for example, 1-1000mg / kg, 1-100mg / kg, 0.5-50mg / kg, 0.1-100mg / kg, 0.5-25mg / kg, 1-20mg / kg and 1-10mg / kg. The exemplary dosage of a composition comprising a binding agent of the present disclosure includes, but is not limited to, 0.1mg / kg, 0.5mg / kg, 1.0mg / kg, 2.0mg / kg, 4mg / kg, 8mg / kg or 20mg / kg.

[0322] Suitable human doses of any composition comprising a binding agent of the present disclosure can be further evaluated in, for example, a Phase I dose escalation study. See, 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.

[0323] In various embodiments, the pharmaceutical composition may comprise a nucleic acid of the present disclosure, such as a vector. Methods for preparing pharmaceutical compositions comprising nucleic acids of the present disclosure are known, such as in the books in the series of Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal or subcutaneous application may comprise the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid, etc.; buffers, such as acetates, citrates or phosphates, and agents for regulating tension, 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 may be packaged in, for example, ampoules, disposable syringes, or vials made of glass or plastic for more than one dose.

[0324] Pharmaceutical compositions suitable for injection comprising nucleic acids of the present disclosure may include sterile aqueous solutions or dispersants and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersants. For intravenous administration, suitable carriers may include physiological saline, antibacterial water, Cremophor EL.TM. (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0325] Sterility, stability, viscosity and other factors relevant to effective therapeutic use can be considered. For example, a method of maintaining fluidity includes using a coating such as lecithin, maintaining the required particle size and using a surfactant. In some cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol, sodium chloride. In some cases, the extended absorption of the injectable composition comprising nucleic acid of the present disclosure can be achieved by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0326] Sterile injectable solutions can be prepared by incorporating one or more ingredients such as antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.) and / or by filtration sterilization. In some cases, dispersions are prepared by incorporating the active molecule into a sterile vehicle containing a basic dispersion medium and an antibacterial or antifungal agent. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying from previously sterile filtered solutions thereof.

[0327] Oral compositions comprising the nucleic acids of the present disclosure may include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the nucleic acids of the present disclosure may be combined with excipients and used in the form of, for example, tablets, lozenges or capsules, such as gelatin capsules. Oral compositions comprising the nucleic acids of the present disclosure may also be prepared using fluid carriers used as mouthwashes. 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 ingredients or molecules of similar nature: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, or corn starch; lubricants such as magnesium stearate or Glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0328] In some embodiments, nucleic acid can be administered by any method suitable for administering nucleic acid agents (such as DNA vaccines). These methods include, for example, gene guns, biological syringes and skin patches and needle-free methods, such as microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and mammalian percutaneous needle-free vaccination using powdered form vaccines disclosed in U.S. Patent No. 6,168,587. In addition, intranasal delivery is possible, as described in Hamajima et al., (1998) Clin. Immunol. Immunopathol. 88 (2), 205-10. Liposomes (for example, as described in U.S. Patent No. 6,472,375). In some cases, microencapsulation can be used. In addition, biodegradable targeted microparticle delivery systems (for example, as described in U.S. Patent No. 6,471,996) can be used.

[0329] Compositions comprising nucleic acid of the present disclosure may include components such as adjuvants, diluents, adhesives, stabilizers, buffers, salts, lipophilic solvents, preservatives, mixtures thereof or any component for inclusion in therapeutic compositions comprising nucleic acid. Nucleic acid compositions may include, for example, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc., phosphate buffered saline, water and emulsions compatible with drug administration, such as oil / water or water / oil emulsions, and various types of wetting agents. Supplementary active molecules may also be incorporated into compositions comprising nucleic acid of the present disclosure. Compositions comprising nucleic acid of the present disclosure of the present disclosure may include stabilizers and preservatives and any carrier as described herein, with the optional additional condition that they are acceptable for use in vivo. For examples of additional 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, NJ (1998).

[0330] 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 route of administration include parenteral, such as intravenous, intracranial, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0331] The nucleic acid composition may include a buffer or a pH regulator. The buffer may be a salt prepared from an organic acid or base. The buffer of the present disclosure includes an organic acid salt, such as a salt of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, Tris, trishydroxymethylaminomethane hydrochloride and a phosphate buffer. Other carriers include polymeric excipients or additives, such as polyvinyl pyrrolidone, polysucrose (a polymeric sugar), dextran (e.g., cyclodextrin, such as 2-hydroxypropyl-ortho-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).

[0332] In some embodiments where the nucleic acid of the present disclosure is a vector, the present disclosure includes compositions for gene transduction and / or gene therapy, for example, compositions comprising viral particles, such as AAV particles and / or retroviruses, such as slow virus particles. As used in reference to viral titers, the term "genomic particles (gp)" or "genomic equivalents" refers to the number of viral particles containing recombinant viral genomes (e.g., recombinant AAV DNA genomes), regardless of infectivity or functionality. The number of genomic particles in a vector preparation can be measured. As used in reference to viral titers, the term "infectious unit (IU)", "infectious particles" or "replication unit" refers to the number of infectivity and replication competent recombinant viruses, such as the number of recombinant AAV vector particles, such as can be measured by an infectious center assay (also referred to as a replication center assay). As used in reference to viral titers, the term "transduction unit (TU)" refers to the number of infectious recombinant vector particles (e.g., recombinant AAV vector particles) that result in the production of functional transgenic products.

[0333] In some embodiments, the composition comprises, for example, 2 x 10 6 Up to 2x10 12 , 2x10 7 Up to 2x10 11 or 2x10 8 Up to 2x10 11 In certain embodiments, the concentration or titer of the vector in a unit dosage form is, for example, at least: (a) 1×10 12 particles / mL, 2x10 12 particles / mL, 3x10 12 particles / mL, 4x10 12 particles / mL, 5x10 12 particles / mL, 6x10 12 particles / mL, 7x1012 particles / mL, 8x10 12 particles / mL, 9x10 12 particles / mL, 10x10 12 particles / mL, 15x10 12 particles / mL, 20x10 12 particles / mL, 25x10 12 particles / mL, or50x10 12 particles / mL; (b) 1x10 9 TU / mL, 2x10 9 TU / mL, 3x10 9 TU / mL, 4x10 9 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, 20x10 10 IU / mL, 25x10 10 IU / mL or 50x10 10 IU / mL. Such embodiments do not limit the unit doses encompassed by the present disclosure and do not limit the various measures of dosage that can be used in conjunction with the various compositions of the present disclosure that contain nucleic acids of the present disclosure. For example, particle dose, concentration, or amount can be measured and / or expressed in terms of vector genomes / kilogram subject (Vg / Kg) or Vg / dose. The preferred manner of measuring and / or expressing particle dose, concentration, or amount can vary depending on various factors, such as route of administration.

[0334] The present disclosure provides techniques for simultaneously targeting CD20 and another antigen (e.g., CD19). In some embodiments, the present disclosure provides techniques for initiating and / or regulating an immune response. In some embodiments, the present disclosure provides techniques for treating cancer (e.g., cancer characterized by cells with surface expressed CD20).

[0335] This specification includes the use of the binding agent pharmaceutical composition provided herein for treating or preventing cancer. Another aspect of the present disclosure relates to a method for treating or preventing a malignant tumor, the method comprising administering an effective amount of a binding agent pharmaceutical composition to a subject in need thereof, for example, wherein the cell comprises at least one antigen binding system provided herein. The method of the present disclosure including administering a pharmaceutically effective amount of a binding agent pharmaceutical composition of the present disclosure can be used to treat cancer in a subject, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from patients, prevent tumor recurrence, prevent tumor metastasis, induce remission in patients, or any combination thereof. In certain embodiments, the method provided herein induces a complete response. In some embodiments, the method provided herein induces a partial response. In certain embodiments, the binding agent pharmaceutical composition is a cell provided herein, comprising a cell provided herein, comprising a cell provided herein as an active agent, or comprising a cell provided herein as the only active agent, the cell, for example, comprising or expressing at least one CAR of the present disclosure. In some embodiments, the binding agent pharmaceutical composition includes a bicistronic CAR system comprising an anti-CD20 CAR and an anti-CD19 CAR, or the binding agent pharmaceutical composition comprises a bispecific anti-C20 / anti-CD19 CAR of the present disclosure.

[0336] In various embodiments, the present disclosure includes the use of the binding agent pharmaceutical composition provided herein to induce or provide immunity to cancer in a subject. The present disclosure further includes a method for preventing cancer in a subject by administering a binding agent pharmaceutical composition provided herein to a subject. The present disclosure further includes a method for inducing an immune response in a subject by administering a binding agent pharmaceutical composition provided herein to a subject. In certain embodiments, the binding agent pharmaceutical composition is a cell provided herein, a cell provided herein, a cell provided herein as an active agent, or a cell provided herein as the only active agent, such as a cell comprising or expressing at least one CAR of the present disclosure. In some embodiments, the binding agent pharmaceutical composition includes a bicistronic CAR system comprising an anti-CD20 CAR and an anti-CD19 CAR, or the binding agent pharmaceutical composition includes a bispecific anti-C20 / anti-CD19 CAR of the present disclosure.

[0337] In certain embodiments, the method for treating a cancer in a subject in need thereof comprises administering to the subject a polynucleotide, vector, antibody, or antigen binding system disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding an antigen binding system or antibody (e.g., an antigen binding system). In another embodiment, the method comprises administering a vector comprising a polynucleotide encoding an antigen binding system or antibody. In another embodiment, the method comprises administering to the subject an antigen binding system or antibody.

[0338] Another aspect of the present disclosure relates to a method for preparing a cell expressing CAR or TCR, comprising transducing a cell with a polynucleotide disclosed herein under suitable conditions. In some embodiments, the method comprises transducing a cell with a polynucleotide encoding CAR or TCR, as disclosed herein. In some embodiments, the method comprises transducing a cell with a vector comprising a polynucleotide encoding CAR or TCR. In certain embodiments, the present disclosure provides a T cell therapy, wherein the binding agent pharmaceutical composition comprises T cells transfected or transduced with a vector, and the vector comprises a polynucleotide sequence encoding an antigen binding agent of the present disclosure (e.g., an antigen binding system). In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for T cell therapy are obtained from a subject who is not a patient. In one embodiment, the T cell therapy of the present disclosure is an autologous cell therapy (eACT TM ). 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 antigen binding system of the present disclosure. In certain embodiments, the binding agent is administered to the patient. In some embodiments, the binding agent treats or is intended to treat a patient's cancer. For example, in one embodiment, the binding agent reduces the size of a tumor. In various embodiments, the cells of the present disclosure 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.

[0339] Another aspect of the present disclosure relates to a method of inducing immunity to a tumor, comprising administering an effective amount of cells to a subject, the cells comprising polynucleotides described herein, vectors described herein, or CAR or TCR described herein. In one embodiment, the method comprises administering an effective amount of cells to a subject, the cells comprising polynucleotides encoding CAR or TCR disclosed herein. In another embodiment, the method comprises administering an effective amount of cells comprising a vector to a subject, the vector comprising polynucleotides encoding CAR or TCR disclosed herein. In another embodiment, the method comprises administering an effective amount of cells to a subject, the cells comprising CAR or TCR encoded by polynucleotides disclosed herein.

[0340] Another aspect of the present disclosure relates to a method of inducing an immune response in a subject, comprising administering an effective amount of an engineered immune cell of the present application. In some embodiments, the immune response is a T cell-mediated immune response. In some embodiments, the T cell-mediated immune response is directed to one or more target cells. In some embodiments, the engineered immune cell comprises a CAR or a TCR, wherein the CAR or the TCR comprises a THD described in the present disclosure. In some embodiments, the target cell is a tumor cell.

[0341] Another aspect of the present disclosure...

Claims

1. An antigen binding system, antibody or antigen binding fragment thereof comprising an anti-CD20 binding motif, wherein the anti-CD20 binding motif comprises the sequence of three heavy chain complementarity determining regions (HCDRs) of any one of the heavy chain variable regions (HCVRs) selected from the group consisting of SEQ ID NOs: 1, 23, 45, 67, 89, 111, 133, 155, 177 and 199, and the sequence of three light chain CDRs (LCDRs) of the light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 12, 34, 56, 78, 100, 122, 144, 166, 188 and 210.

2. The antigen binding system, antibody or antigen binding fragment thereof of claim 1, wherein the anti-CD20 binding motif comprises a first domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and a second domain comprising three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein (i) the HCDR1 has a sequence according to any one of SEQ ID NOs: 3-5, 25-27, 47-49, 69-71, 91-93, 113-115, 135-137, 157-159, 179-181; and 201-203; (ii) the HCDR2 has a sequence according to any one of SEQ ID NOs: 6-8, 28-30, 50-52, 72-74, 94-96, 116-118, 138-140, 160-162, 182-184; and 204-206; (iii) the HCDR3 has a sequence according to any one of SEQ ID NOs: 9-11, 31-33, 53-55, 75-77, 97-99, 119-121, 141-143, 163-165, 185-187; and 207-209; (iv) the LCDR1 has a sequence according to any one of SEQ ID NOs: 14-16, 36-38, 58-60, 80-82, 102-104, 124-126, 146-148, 168-170, 190-192; and 212-214; (v) the LCDR2 has a sequence according to any one of SEQ ID NOs: 17-19, 39-41, 61-63, 83-85, 105-107, 127-129, 149-151, 171-173, 193-195; and 215-217; and (vi) the LCDR3 has a sequence according to any one of SEQ ID NOs: 20-22, 42-44, 64-66, 86-88, 108-110, 130-132, 152-154, 174-176, 196-198; and 218-220.

3. The antigen binding system, antibody or antigen binding fragment thereof of claim 1 or 2, wherein the HCDR comprises: (i) a HCDR1 according to any one of SEQ ID NOs: 3-5; a HCDR2 according to any one of SEQ ID NOs: 6-8; a HCDR3 according to any one of SEQ ID NOs: 9-11; (ii) a HCDR1 according to any one of SEQ ID NOs: 25-27; a HCDR2 according to any one of SEQ ID NOs: 28-30; a HCDR3 according to any one of SEQ ID NOs: 31-33; (iii) a HCDR1 according to any one of SEQ ID NOs: 47-49; a HCDR2 according to any one of SEQ ID NOs: 50-52; a HCDR3 according to any one of SEQ ID NOs: 53-55; (iv) a HCDR1 according to any one of SEQ ID NOs: 69-71; a HCDR2 according to any one of SEQ ID NOs: 72-74; a HCDR3 according to any one of SEQ ID NOs: 75-77; (v) a HCDR1 according to any one of SEQ ID NOs: 91-93; a HCDR2 according to any one of SEQ ID NOs: 94-96; a HCDR3 according to any one of SEQ ID NOs: 97-99; (vi) a HCDR1 according to any one of SEQ ID NOs: 113-115; a HCDR2 according to any one of SEQ ID NOs: 116-118; a HCDR3 according to any one of SEQ ID NOs: 119-121; (vii) a HCDR1 according to any one of SEQ ID NOs: 135-137; a HCDR2 according to any one of SEQ ID NOs: 138-140; a HCDR3 according to any one of SEQ ID NOs: 141-143; (viii) a HCDR1 according to any one of SEQ ID NOs: 157-159; a HCDR2 according to any one of SEQ ID NOs: 160-162; a HCDR3 according to any one of SEQ ID NOs: 163-165; (ix) a HCDR1 according to any one of SEQ ID NOs: 179-181; a HCDR2 according to any one of SEQ ID NOs: 182-184; a HCDR3 according to any one of SEQ ID NOs: 185-187; or (x) a HCDR1 according to any one of SEQ ID NOs: 201-203; a HCDR2 according to any one of SEQ ID NOs: 204-206; a HCDR3 according to any one of SEQ ID NOs: 207-209; and The LCDR 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 LCDR2 according to any one of SEQ ID NO:61 - 63; an LCDR3 according to any one of SEQ ID NO:64 - 66; (iv) An LCDR1 according to any one of SEQ ID NO:80 - 82; an LCDR2 according to any one of SEQ ID NO:83 - 85; an LCDR3 according to any one of SEQ ID NO:86 - 88; (v) An LCDR1 according to any one of SEQ ID NO:102 - 104; an LCDR2 according to any one of SEQ ID NO:105 - 107; an LCDR3 according to any one of SEQ ID NO:108 - 110; (vi) An LCDR1 according to any one of SEQ ID NO:124 - 126; an LCDR2 according to any one of SEQ ID NO:127 - 129; an LCDR3 according to any one of SEQ ID NO:130 - 132; (vii) An LCDR1 according to any one of SEQ ID NO:146 - 148; an LCDR2 according to any one of SEQ ID NO:149 - 151; an LCDR3 according to any one of SEQ ID NO:152 - 154; (viii) An LCDR1 according to any one of SEQ ID NO:168 - 170; an LCDR2 according to any one of SEQ ID NO:171 - 173; an LCDR3 according to any one of SEQ ID NO:174 - 176; (ix) An LCDR1 according to any one of SEQ ID NO:190 - 192; an LCDR2 according to any one of SEQ ID NO:193 - 195; an LCDR3 according to any one of SEQ ID NO:196 - 198; or (x) An LCDR1 according to any one of SEQ ID NO:212 - 214; an LCDR2 according to any one of SEQ ID NO:215 - 217; an LCDR3 according to any one of SEQ ID NO:218 - 220.

4. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1 to 3, wherein the antigen binding system, antibody or antigen binding fragment thereof comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), in: The HCDR and LCDR comprise: (i) a HCDR1 according to any one of SEQ ID NOs: 3-5; a HCDR2 according to any one of SEQ ID NOs: 6-8; a HCDR3 according to any one of SEQ ID NOs: 9-11; a LCDR1 according to any one of SEQ ID NOs: 14-16; a LCDR2 according to any one of SEQ ID NOs: 17-19; a LCDR3 according to any one of SEQ ID NOs: 20-22; (ii) a HCDR1 according to any one of SEQ ID NOs: 25-27; a HCDR2 according to any one of SEQ ID NOs: 28-30; a HCDR3 according to any one of SEQ ID NOs: 31-33; a LCDR1 according to any one of SEQ ID NOs: 36-38; a LCDR2 according to any one of SEQ ID NOs: 39-41; a LCDR3 according to any one of SEQ ID NOs: 42-44; (iii) a HCDR1 according to any one of SEQ ID NOs: 47-49; a HCDR2 according to any one of SEQ ID NOs: 50-52; a HCDR3 according to any one of SEQ ID NOs: 53-55; a LCDR1 according to any one of SEQ ID NOs: 58-60; a LCDR2 according to any one of SEQ ID NOs: 61-63; a LCDR3 according to any one of SEQ ID NOs: 64-66; (iv) a HCDR1 according to any one of SEQ ID NOs: 69-71; a HCDR2 according to any one of SEQ ID NOs: 72-74; a HCDR3 according to any one of SEQ ID NOs: 75-77; a LCDR1 according to any one of SEQ ID NOs: 80-82; a LCDR2 according to any one of SEQ ID NOs: 83-85; a LCDR3 according to any one of SEQ ID NOs: 86-88; (v) a HCDR1 according to any one of SEQ ID NOs: 91-93; a HCDR2 according to any one of SEQ ID NOs: 94-96; a HCDR3 according to any one of SEQ ID NOs: 97-99; a LCDR1 according to any one of SEQ ID NOs: 102-104; a LCDR2 according to any one of SEQ ID NOs: 105-107; a LCDR3 according to any one of SEQ ID NOs: 108-110; (vi) An HCDR1 according to any one of SEQ ID NOs: 113 - 115; an HCDR2 according to any one of SEQ ID NOs: 116 - 118; an HCDR3 according to any one of SEQ ID NOs: 119 - 121; an LCDR1 according to any one of SEQ ID NOs: 124 - 126; an LCDR2 according to any one of SEQ ID NOs: 127 - 129; an LCDR3 according to any one of SEQ ID NOs: 130 - 132; (vii) An HCDR1 according to any one of SEQ ID NOs: 135 - 137; an HCDR2 according to any one of SEQ ID NOs: 138 - 140; an HCDR3 according to any one of SEQ ID NOs: 141 - 143; an LCDR1 according to any one of SEQ ID NOs: 146 - 148; an LCDR2 according to any one of SEQ ID NOs: 149 - 151; an LCDR3 according to any one of SEQ ID NOs: 152 - 154; (viii) An HCDR1 according to any one of SEQ ID NOs: 157 - 159; an HCDR2 according to any one of SEQ ID NOs: 160 - 162; an HCDR3 according to any one of SEQ ID NOs: 163 - 165; an LCDR1 according to any one of SEQ ID NOs: 168 - 170; an LCDR2 according to any one of SEQ ID NOs: 171 - 173; an LCDR3 according to any one of SEQ ID NOs: 174 - 176; (ix) An HCDR1 according to any one of SEQ ID NOs: 179 - 181; an HCDR2 according to any one of SEQ ID NOs: 182 - 184; an HCDR3 according to any one of SEQ ID NOs: 185 - 187; an LCDR1 according to any one of SEQ ID NOs: 190 - 192; an LCDR2 according to any one of SEQ ID NOs: 193 - 195; an LCDR3 according to any one of SEQ ID NOs: 196 - 198; or (x) An HCDR1 according to any one of SEQ ID NOs: 201 - 203; an HCDR2 according to any one of SEQ ID NOs: 204 - 206; an HCDR3 according to any one of SEQ ID NOs: 207 - 209; an LCDR1 according to any one of SEQ ID NOs: 212 - 214; an LCDR2 according to any one of SEQ ID NOs: 215 - 217; an LCDR3 according to any one of SEQ ID NOs: 218 - 220.

5. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1 to 4, wherein the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs, in: (i) the heavy chain variable domain is at least 80% identical to 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: 177 or SEQ ID NO: 199; and (ii) the light chain variable 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.

6. The antigen binding system, antibody or antigen binding fragment thereof of claim 5, wherein the antigen binding system, antibody or antigen binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs, in: (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1, and the light chain variable domain is at least 80% identical to SEQ ID NO: 12; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:23, and the light chain variable domain is at least 80% identical to SEQ ID NO:34; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO:45, and the light chain variable domain is at least 80% identical to SEQ ID NO:56; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO:67, and the light chain variable domain is at least 80% identical to SEQ ID NO:78; (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO:89, and the light chain variable domain is at least 80% identical to SEQ ID NO:100; (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 111, and the light chain variable domain is at least 80% identical to SEQ ID NO: 122; (vii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 133, and the light chain variable domain is at least 80% identical to SEQ ID NO: 144; (viii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 155, and the light chain variable domain is at least 80% identical to SEQ ID NO: 166; (ix) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 177, and the light chain variable domain is at least 80% identical to SEQ ID NO: 188; or (x) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 199, and the light chain variable domain is at least 80% identical to SEQ ID NO:

210.

7. The antigen binding system, antibody or antigen binding fragment thereof according to any one of claims 1 to 6, wherein the three HCDRs and the three LCDRs are comprised by a single polypeptide.

8. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1 to 6, wherein the three HCDRs are comprised by a first polypeptide and the three LCDRs are comprised by a second polypeptide.

9. The antigen binding system, antibody or antigen binding fragment thereof of claim 8, wherein the first polypeptide is an antibody heavy chain and the second polypeptide is an antibody light chain.

10. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1 to 9, wherein the antigen binding system, antibody or antigen binding fragment thereof further comprises: (i) a binding motif that specifically binds to an antigen selected from the group consisting of 5T4, alpha-fetoprotein, B cell maturation antigen (BCMA), B cell receptor, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, 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 gpl20, IL-1Ralpha, kappa chain, lambda chain, melanoma associated antigen, mesothelin, MUC-1, mutated p53, mutated ras, prostate specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B cell activating factor receptor), and combinations thereof; and / or (ii) a binding motif that specifically binds to a B-cell characteristic antigen, optionally wherein the B-cell characteristic antigen is not CD19 or CD20.

11. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 1 to 9, wherein the antigen binding system, antibody or antigen binding fragment thereof further comprises an anti-CD19 binding motif.

12. The antigen binding system, antibody or antigen binding fragment thereof of claim 11, wherein the anti-CD19 binding motif comprises a first domain comprising three HCDRs and a second domain comprising three LCDRs, in: The three HCDRs of the anti-CD19 binding motif include HCDR1, HCDR2 and HCDR3; The three LCDRs of the anti-CD19 binding motif include LCDR1, LCDR2 and LCDR3; and The HCDR and LCDR of the anti-CD19 binding motif comprise HCDR1 according to any one of SEQ ID NOs: 223-225; HCDR2 according to any one of SEQ ID NOs: 226-228; HCDR3 according to any one of SEQ ID NOs: 229-231; LCDR1 according to any one of SEQ ID NOs: 234-236; LCDR2 according to any one of SEQ ID NOs: 237-239; LCDR3 according to any one of SEQ ID NOs: 240-242.

13. The antigen binding system, antibody or antigen binding fragment thereof of claim 12, wherein the anti-CD19 binding motif comprises a first heavy chain variable domain comprising the three HCDRs of the anti-CD19 binding motif and a light chain variable domain comprising the 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.

14. The antigen binding system, antibody or antigen binding fragment thereof of claim 12 or claim 13, wherein the three HCDRs of the anti-CD19 binding motif and the three LCDRs of the anti-CD19 binding motif are comprised by a single polypeptide.

15. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 12-14, wherein the three HCDRs of the anti-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 together comprised by a single polypeptide.

16. The antigen binding system, antibody or antigen binding fragment thereof according to any one of claims 1 to 15, wherein the antigen binding system, antibody or antigen binding fragment thereof is or is comprised of a chimeric antigen receptor.

17. The antigen binding system, antibody or antigen binding fragment thereof of claim 16, wherein the chimeric antigen receptor comprises a transmembrane domain which is a transmembrane domain of 4-1BB / CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154 or a zeta chain of a T cell receptor, or any combination thereof.

18. The antigen binding system, antibody or antigen binding fragment thereof of any one of claims 11 to 14, wherein (i) the three HCDRs of the anti-CD20 binding motif and the three LCDRs of the anti-CD20 binding motif are present in a 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 comprised by a second different polypeptide.

19. The antigen binding system, antibody or antigen binding fragment thereof of claim 18, wherein the first polypeptide is or is comprised by a first chimeric antigen receptor.

20. The antigen binding system, antibody or antigen binding fragment thereof of claim 18 or claim 19, wherein the second polypeptide is or is comprised by a second chimeric antigen receptor.

21. A nucleic acid encoding at least one polypeptide according to any one of claims 1 to 20.

22. A vector comprising the nucleic acid of claim 21.

23. A method for producing an engineered cell, the method comprising transfecting or transducing the cell with a nucleic acid according to claim 21 or a vector according to claim 22.

24. A cell encoding or expressing the antigen binding system, antibody or antigen binding fragment thereof according to any one of claims 1 to 20, optionally wherein the cell is an immune cell, optionally wherein the cell is a T cell.

25. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising the antigen binding system, antibody, or antigen binding fragment thereof of any one of claims 1-20.

26. A method of inducing an immune response in a subject or immunizing a subject against cancer, the method comprising administering to the subject a cell therapy composition comprising one or more cells encoding or comprising the antigen binding system, antibody or antigen binding fragment of any one of claims 1-20.

27. The method of claim 25 or 26, wherein the cell is a CAR-T cell.

28. The method of any one of claims 25-27, wherein the cancer is acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasms, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic 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 conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (including non- Symptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma or Waldenstrom's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, hairy cell lymphoma / leukemia, or a combination thereof.

29. The method of any one of claims 25-28, wherein the cell therapy is allogeneic cell therapy or autologous cell therapy.

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