Chimeric antigen and t cell receptors and methods of use

Abstract

To provide a chimeric antigen receptor (CAR) and an engineered T cell receptor (TCR) to interact with specific tumor antigens and to target and kill cancer cells.SOLUTION: Provided are antibodies and antigen-binding systems comprising a binding motif that binds to CD20 and, optionally, a binding motif that binds to CD19, having specific sequences, as well as methods for producing and using the same. Antibodies and antigen binding systems of the present disclosure comprise CARs that comprise an anti-CD20 binding motif and an anti-CD19 binding motif. Compositions, such as antibodies and CARs that are or comprise an anti-CD20 / anti-CD19 antigen binding system of the present disclosure, are provided and cell therapies comprising the composition are useful, e.g., in the treatment of cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 778,893, filed December 12, 2018, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Human cancers are essentially composed of normal cells that undergo genetic or epigenetic transformation to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that are distinctly different 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 utilize various mechanisms to prevent immune cells, such as T lymphocytes and B lymphocytes, from successfully targeting the cancer cells.

[0003] Current therapeutic T cell therapy relies on enriched or engineered human T cells to target and kill cancer cells in patients. 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 contain binding domains that can interact with specific tumor antigens, enable T cells to target and kill cancer cells that express specific tumor antigens. CARs and TCRs are needed to target and kill cancer cells. Summary of the Invention

[0004] In at least a first aspect, the present disclosure comprises 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 heavy chain variable regions (HCVRs) selected from the group consisting of 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, and SEQ ID NO:199, and the sequence of three light chain CDRs (LCDRs) of any one of light chain variable regions (LCVRs) selected from the group consisting of 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, and SEQ ID NO:210. In some embodiments, 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 to 5, 25 to 27, 47 to 49, 69 to 71, 91 to 93, 113 to 115, 135 to 137, 157 to 159, 179 to 181, and 201 to 203; and (ii) the HCDR2 has a sequence according to any one of SEQ ID NOs: 3 to 5, 25 to 27, 47 to 49, 69 to 71, 91 to 93, 113 to 115, 135 to 137, 157 to 159, 179 to 181, and 201 to 203. (iii) the HCDR3 has a sequence according to any one of SEQ ID NOs: 9 to 11, SEQ ID NOs: 31 to 33, SEQ ID NOs: 53 to 55, SEQ ID NOs: 75 to 77, SEQ ID NOs: 97 to 99, SEQ ID NOs: 119 to 121, SEQ ID NOs: 141 to 143, SEQ ID NOs: 163 to 165, SEQ ID NOs: 185 to 187, and the sequence(iv) the LCDR1 has a sequence according to any one of SEQ ID NOs: 14 to 16, 36 to 38, 58 to 60, 80 to 82, 102 to 104, 124 to 126, 146 to 148, 168 to 170, 190 to 192, and 212 to 214; and (v) the LCDR2 has a sequence according to any one of SEQ ID NOs: 17 to 19, 39 to 41, 61 to 63, 83 to 85, 105 to 110 and (vi) the LCDR3 has a sequence according to any one of SEQ ID NOs: 20 to 22, 42 to 44, 64 to 66, 86 to 88, 108 to 110, 130 to 132, 152 to 154, 174 to 176, 196 to 198, and 218 to 220. In some embodiments, the HCDR is (i) an HCDR1 according to any one of SEQ ID NOs: 3 to 5, an HCDR2 according to any one of SEQ ID NOs: 6 to 8, and an HCDR3 according to any one of SEQ ID NOs: 9 to 11; (ii) an HCDR1 according to any one of SEQ ID NOs: 25 to 27, an HCDR2 according to any one of SEQ ID NOs: 28 to 30, and an HCDR3 according to any one of SEQ ID NOs: 31 to 33; (iii) an HCDR1 according to any one of SEQ ID NOs: 47 to 49, an HCDR2 according to any one of SEQ ID NOs: 50 to 52, and an HCDR3 according to any one of SEQ ID NOs: 53 to 55; (iv) an HCDR1 according to any one of SEQ ID NOs: 69 to 71, an HCDR2 according to any one of SEQ ID NOs: 72 to 74, and an HCDR3 according to any one of SEQ ID NOs: 75 to 77; (v) an HCDR1 according to any one of SEQ ID NOs: 91 to 93, and an HCDR2 according to any one of SEQ ID NOs: 94 to 96;(vi) an HCDR1 according to any one of SEQ ID NOs: 113 to 115, an HCDR2 according to any one of SEQ ID NOs: 116 to 118, and an HCDR3 according to any one of SEQ ID NOs: 119 to 121, (vii) an HCDR1 according to any one of SEQ ID NOs: 135 to 137, an HCDR2 according to any one of SEQ ID NOs: 138 to 140, and an HCDR3 according to any one of SEQ ID NOs: 141 to 143, (viii) an HCDR according to any one of SEQ ID NOs: 157 to 159 CDR1, an HCDR2 according to any one of SEQ ID NOs: 160 to 162, an HCDR3 according to any one of SEQ ID NOs: 163 to 165, (ix) an HCDR1 according to any one of SEQ ID NOs: 179 to 181, an HCDR2 according to any one of SEQ ID NOs: 182 to 184, an HCDR3 according to any one of SEQ ID NOs: 185 to 187, or (x) an HCDR1 according to any one of SEQ ID NOs: 201 to 203, an HCDR2 according to any one of SEQ ID NOs: 204 to 206, an HCDR3 according to any one of SEQ ID NOs: 207 to 209 and the LCDR comprises an HCDR3, and the LCDR is (i) an LCDR1 according to any one of SEQ ID NOs: 14 to 16, an LCDR2 according to any one of SEQ ID NOs: 17 to 19, or an LCDR3 according to any one of SEQ ID NOs: 20 to 22; (ii) an LCDR1 according to any one of SEQ ID NOs: 36 to 38, an LCDR2 according to any one of SEQ ID NOs: 39 to 41, or an LCDR3 according to any one of SEQ ID NOs: 42 to 44; or (iii) an LCDR1 according to any one of SEQ ID NOs: 58 to 60, or an LCDR2 according to any one of SEQ ID NOs: 61 to 63. (iv) LCDR1 according to any one of SEQ ID NOs: 80 to 82, LCDR2 according to any one of SEQ ID NOs: 83 to 85, LCDR3 according to any one of SEQ ID NOs: 86 to 88, (v) LCDR1 according to any one of SEQ ID NOs: 102 to 104, LCDR2 according to any one of SEQ ID NOs: 105 to 107, LCDR3 according to any one of SEQ ID NOs: 108 to 110, (vi) LCDR1 according to any one of SEQ ID NOs: 124 to 126,SEQ ID NO: 127 to SEQ ID NO: 129 (vii) an LCDR1 according to any of SEQ ID NOs: 146 to 148, an LCDR2 according to any of SEQ ID NOs: 149 to 151, and an LCDR3 according to any one of SEQ ID NOs: 152 to 154, (viii) an LCDR1 according to any of SEQ ID NOs: 168 to 170, an LCDR2 according to any of SEQ ID NOs: 171 to 173, and an LCDR3 according to any one of SEQ ID NOs: 174 to 176, (ix) an LCDR1 according to any of SEQ ID NOs: 190 to 192, an LCDR2 according to any of SEQ ID NOs: 193 to 195, and an LCDR3 according to any one of SEQ ID NOs: 196 to 198, or (x) an LCDR1 according to any of SEQ ID NOs: 212 to 214, an LCDR2 according to any of SEQ ID NOs: 215 to 217, and an LCDR3 according to any one of SEQ ID NOs: 218 to 220.

[0005] In various embodiments, the antigen binding system, antibody, or antigen-binding fragment thereof of the present disclosure comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), wherein the HCDRs and the LCDRs are selected from the group consisting of: (i) an HCDR1 according to any one of SEQ ID NOs: 3 to 5, an HCDR2 according to any one of SEQ ID NOs: 6 to 8, an HCDR3 according to any one of SEQ ID NOs: 9 to 11, an LCDR1 according to any one of SEQ ID NOs: 14 to 16, an LCDR2 according to any one of SEQ ID NOs: 17 to 18, an LCDR3 according to any one of SEQ ID NOs: 19 to 20, an LCDR4 according to any one of SEQ ID NOs: 21 to 23, an LCDR5 according to any one of SEQ ID NOs: 24 to 25, an LCDR6 according to any one of SEQ ID NOs: 26 to 27, an LCDR7 according to any one of SEQ ID NOs: 28 to 29, an LCDR8 according to any one of SEQ ID NOs: 30 to 31, an LCDR9 according to any one of SEQ ID NOs: 32 to 33, an LCDR10 according to any one of SEQ ID NOs: 34 to 35, an LCDR11 according to any one of SEQ ID NOs: 36 to 37, an LCDR12 according to any one of SEQ ID NOs: 38 to 39, an LCDR13 according to any one of SEQ ID NOs: 39 to 40, an LCDR14 according to any one of SEQ ID NOs: 41 to 42, an LCDR15 according to any one of SEQ ID NOs: 43 to 44, an LCDR16 according to any one of SEQ ID NOs: 9, and LCDR3 according to any one of SEQ ID NOs: 20 to 22; (ii) HCDR1 according to any one of SEQ ID NOs: 25 to 27, HCDR2 according to any one of SEQ ID NOs: 28 to 30, HCDR3 according to any one of SEQ ID NOs: 31 to 33, LCDR1 according to any one of SEQ ID NOs: 36 to 38, LCDR2 according to any one of SEQ ID NOs: 39 to 41, and LCDR3 according to any one of SEQ ID NOs: 42 to 44; (iii) HCDR1 according to any one of SEQ ID NOs: 47 to 49, HCDR2 according to any one of SEQ ID NOs: 50 to 52, HCDR3 according to any one of SEQ ID NOs: 53 to 55, LCDR1 according to any one of SEQ ID NOs: 58 to 60, LCDR2 according to any one of SEQ ID NOs: 61 to 63, and LCDR3 according to any one of SEQ ID NOs: 64 to 66; (iv) HCDR1 according to any one of SEQ ID NOs: 69 to 71, HCDR2 according to any one of SEQ ID NOs: 72 to 74, and HCDR3 according to any one of SEQ ID NOs: 75 to 77, and (v) an HCDR1 according to any one of SEQ ID NOs: 91 to 93, an HCDR2 according to any one of SEQ ID NOs: 94 to 96, an HCDR3 according to any one of SEQ ID NOs: 97 to 99, an LCDR1 according to any one of SEQ ID NOs: 102 to 104, an LCDR2 according to any one of SEQ ID NOs: 105 to 107, and an LCDR3 according to any one of SEQ ID NOs: 108 to 110,(vi) HCDR1 according to any one of SEQ ID NOs: 113 to 115, HCDR2 according to any one of SEQ ID NOs: 116 to 118, HCDR3 according to any one of SEQ ID NOs: 119 to 121, LCDR1 according to any one of SEQ ID NOs: 124 to 126, LCDR2 according to any one of SEQ ID NOs: 127 to 129, LCDR3 according to any one of SEQ ID NOs: 130 to 132, (vii) HCDR1 according to any one of SEQ ID NOs: 135 to 137, HCDR2 according to any one of SEQ ID NOs: 138 to 140, HCDR according to any one of SEQ ID NOs: 141 to 143 R3, an LCDR1 according to any one of SEQ ID NOs: 146 to 148, an LCDR2 according to any one of SEQ ID NOs: 149 to 151, and an LCDR3 according to any one of SEQ ID NOs: 152 to 154, (viii) an HCDR1 according to any one of SEQ ID NOs: 157 to 159, an HCDR2 according to any one of SEQ ID NOs: 160 to 162, an HCDR3 according to any one of SEQ ID NOs: 163 to 165, an LCDR1 according to any one of SEQ ID NOs: 168 to 170, an LCDR2 according to any one of SEQ ID NOs: 171 to 173, and an LCDR according to any one of SEQ ID NOs: 174 to 176, R3, (ix) an HCDR1 according to any of SEQ ID NOs: 179 to 181, an HCDR2 according to any of SEQ ID NOs: 182 to 184, an HCDR3 according to any one of SEQ ID NOs: 185 to 187, an LCDR1 according to any of SEQ ID NOs: 190 to 192, an LCDR2 according to any of SEQ ID NOs: 193 to 195, an LCDR3 according to any one of SEQ ID NOs: 196 to 198, or (x) an HCDR1 according to any of SEQ ID NOs: 201 to 203, an HCDR2 according to any of SEQ ID NOs: 204 to 206, an HCDR3 according to any one of SEQ ID NOs: 207 to 209, an LCDR1 according to any of SEQ ID NOs: 212 to 214, an LCDR2 according to any of SEQ ID NOs: 215 to 217, and an LCDR3 according to any one of SEQ ID NOs: 218 to 220. In some embodiments, the antigen binding system, antibody, or antigen-binding fragment thereof comprises a first heavy chain variable domain comprising the three HCDRs and a light chain variable domain comprising the three LCDRs, wherein (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO:1, 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 the three HCDRs and a light chain variable domain comprising the three LCDRs, wherein (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1 and the light chain variable domain is at least 80% identical to SEQ ID NO: 12; (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 23 and the light chain variable domain is at least 80% identical to SEQ ID NO: 34; (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 45 and the light chain variable domain is at least 80% identical to SEQ ID NO: 56; (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 67 and the light chain variable domain is at least 80% identical to SEQ ID NO: 78; and (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 89. (vi) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 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.

[0006] In some embodiments of the present disclosure comprising three HCDRs and three LCDRs, the three HCDRs and 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.

[0007] In some embodiments, the antigen binding system, antibody, or antigen-binding fragment thereof is selected from the group consisting of: (i) 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, and / or (ii) a binding motif that binds to an antigen selected from the group consisting of CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, the HER1-HER2 combination, the HER2-HER3 combination, HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Rα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B-cell activating factor receptor), and combinations thereof, wherein optionally, 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, and 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 of SEQ ID NOs: 223 to 225, HCDR2 according to any of SEQ ID NOs: 226 to 228, HCDR3 according to any one of SEQ ID NOs: 229 to 231, LCDR1 according to any of SEQ ID NOs: 234 to 236, LCDR2 according to any of SEQ ID NOs: 237 to 239, and LCDR3 according to any one of SEQ ID NOs: 240 to 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 in a single polypeptide. In some embodiments, the three HCDRs of the anti-CD20 binding motif, the three LCDRs of the anti-CD20 binding motif, the three HCDRs of the anti-CD19 binding motif, and the three LCDRs of the anti-CD19 binding motif are together comprised in a single polypeptide.

[0008] In various embodiments, the antigen binding system, antibody, or antigen-binding fragment thereof is or is comprised in a chimeric antigen receptor. In some embodiments, the antigen binding system, antibody, or antigen-binding fragment thereof is or is a single polypeptide comprised in a chimeric antigen receptor, which is a bispecific chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that is 4-1BB / CD137, a T cell receptor alpha chain, a T cell receptor beta chain, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a T cell receptor zeta chain, 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 in a second, different polypeptide. In some embodiments, the first polypeptide is or is comprised in a first chimeric antigen receptor. In some embodiments, the second polypeptide is or is comprised in a second chimeric antigen receptor.

[0009] In various embodiments, the present disclosure includes nucleic acids encoding at least one polypeptide of the present disclosure and / or vectors comprising such nucleic acids. Additionally, the present disclosure includes methods of making engineered cells, comprising transfecting or transducing a cell with a nucleic acid encoding at least one polypeptide of the present disclosure. Also provided herein are cells that encode or express an antigen binding system, antibody, or antigen-binding fragment provided herein, optionally wherein the cell is an immune cell, and optionally wherein the cell is a T cell.

[0010] The present disclosure further includes a method of treating cancer in a subject in need thereof, 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 of eliciting an immune response in a subject or immunizing a subject against cancer, 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 cells are CAR-T cells. In various embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, 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 syndromes, non-Hodgkin's lymphoma (NHL), and thyroid cancer. The cell therapy may be a plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including dysplasmocyte proliferation; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or Waldenstrom's macroglobulinemia, or a combination thereof. In some embodiments, the cell therapy is allogeneic cell therapy or autologous cell therapy.

[0011] At least one embodiment of the present disclosure includes a chimeric antigen receptor comprising an anti-CD20 binding motif, wherein the anti-CD20 binding motif comprises three heavy chain complementarity determining regions (HCDRs) comprised in any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of 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, and SEQ ID NO: 199, and three light chain CDRs (LCDRs) comprised in any one of the light chain variable region (LCVR) sequences selected from the group consisting of 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, and SEQ ID NO: 210. In some embodiments, in a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), (i) the HCDR1 has a sequence according to any one of SEQ ID NOs: 3 to 5, 25 to 27, 47 to 49, 69 to 71, 91 to 93, 113 to 115, 135 to 137, 157 to 159, 179 to 181, and 201 to 203, and (ii) the HCDR2 has a sequence according to any one of SEQ ID NOs: 6 to 8, 28 to 30, 50 to 52, 72 to 74, 94 to 96, or 100. (iii) the HCDR3 has any one of SEQ ID NOs: 9 to 11, 31 to 33, 53 to 55, 75 to 77, 97 to 99, 119 to 121, 141 to 144, and 204 to 206; and (iv) the LCDR1 has a sequence according to any one of SEQ ID NOs: 14 to 16, SEQ ID NOs: 36 to 38, SEQ ID NOs: 58 to 60, SEQ ID NOs: 80 to 82, SEQ ID NOs: 102 to 104, SEQ ID NOs: 124 to 126, SEQ ID NOs: 146 to 148, SEQ ID NOs: 168 to 170, SEQ ID NOs: (v) the LCDR2 has a sequence according to any one of SEQ ID NOs: 17 to 19, SEQ ID NOs: 39 to 41, SEQ ID NOs: 61 to 63, SEQ ID NOs: 83 to 85, SEQ ID NOs: 105 to 107, SEQ ID NOs: 127 to 129, SEQ ID NOs: 149 to 151, SEQ ID NOs: 171 to 173, SEQ ID NOs: 193 to 195, and SEQ ID NOs: 215 to 216; and (vi) the LCDR3 has a sequence according to any one of SEQ ID NOs: 20 to 22, 42 to 44, 64 to 66, 86 to 88, 108 to 110, 130 to 132, 152 to 154, 174 to 176, 196 to 198, and 218 to 220. In some embodiments, the HCDRs are (i) an HCDR1 according to any one of SEQ ID NOs: 3 to 5, an HCDR2 according to any one of SEQ ID NOs: 6 to 8, and an HCDR3 according to any one of SEQ ID NOs: 9 to 11, (ii) an HCDR1 according to any one of SEQ ID NOs: 25 to 27, and an HCDR2 according to any one of SEQ ID NOs: 28 to 30,(iii) an HCDR1 according to any one of SEQ ID NOs: 47 to 49, an HCDR2 according to any one of SEQ ID NOs: 50 to 52, and an HCDR3 according to any one of SEQ ID NOs: 53 to 55, (iv) an HCDR1 according to any one of SEQ ID NOs: 69 to 71, an HCDR2 according to any one of SEQ ID NOs: 72 to 74, and an HCDR3 according to any one of SEQ ID NOs: 75 to 77, (v) an HCDR1 according to any one of SEQ ID NOs: 91 to 93, an HCDR2 according to any one of SEQ ID NOs: 94 to 95, and an HCDR3 according to any one of SEQ ID NOs: 96 to 97. (vi) an HCDR1 according to any of SEQ ID NOs: 113 to 115, an HCDR2 according to any of SEQ ID NOs: 116 to 118, and an HCDR3 according to any of SEQ ID NOs: 119 to 121; (vii) an HCDR1 according to any of SEQ ID NOs: 135 to 137, an HCDR2 according to any of SEQ ID NOs: 138 to 140, and an HCDR3 according to any of SEQ ID NOs: 141 to 143; (viii) an HCDR1 according to any of SEQ ID NOs: 15 (ix) an HCDR1 according to any one of SEQ ID NOs: 179 to 181, an HCDR2 according to any one of SEQ ID NOs: 182 to 184, an HCDR3 according to any one of SEQ ID NOs: 185 to 187, or (x) an HCDR1 according to any one of SEQ ID NOs: 201 to 203, an HCDR2 according to any one of SEQ ID NOs: 204 to 206, an HCDR3 according to any one of SEQ ID NOs: 207 to 209, or (x) an HCDR1 according to any one of SEQ ID NOs: 208 to 209, an HCDR2 according to any one of SEQ ID NOs: 210 to 211, an HCDR2 according to any one of SEQ ID NOs: 211 to 212, an HCDR3 according to any one of SEQ ID NOs: 212 to 213, an HCDR2 according to any one of SEQ ID NOs: 213 to 214, an HCDR3 according to any one of SEQ ID NOs: 214 to 215, an HCDR1 according to any one of SEQ ID NOs: 215 to 216, an HCDR2 according to any one of SEQ ID NOs: 216 to 217, an HCDR2 according to any one of SEQ ID NOs: 217 to 218, an HCDR3 according to any one of SEQ ID NOs: 218 to 219, an HCDR1 according to any one of SEQ ID NOs: 219 to 220, an HCDR2 according to any one of SEQ ID NOs: 220 to 221, an HCDR2 according to any one of SEQ ID NOs: 221 to 222, an HCDR2 according and an HCDR3 according to any one of SEQ ID NOs: 07 to 209, and the LCDR is selected from the group consisting of (i) an LCDR1 according to any one of SEQ ID NOs: 14 to 16, an LCDR2 according to any one of SEQ ID NOs: 17 to 19, and an LCDR3 according to any one of SEQ ID NOs: 20 to 22, (ii) an LCDR1 according to any one of SEQ ID NOs: 36 to 38, an LCDR2 according to any one of SEQ ID NOs: 39 to 41, and an LCDR3 according to any one of SEQ ID NOs: 42 to 44, (iii) an LCDR1 according to any one of SEQ ID NOs: 58 to 60,LCDR2 according to any one of SEQ ID NO: 61 to SEQ ID NO: 63, SEQ ID NO: 64 to SEQ ID NO: 66, (iv) an LCDR1 according to any one of SEQ ID NOs: 80 to 82, an LCDR2 according to any one of SEQ ID NOs: 83 to 85, and an LCDR3 according to any one of SEQ ID NOs: 86 to 88, (v) an LCDR1 according to any one of SEQ ID NOs: 102 to 104, an LCDR2 according to any one of SEQ ID NOs: 105 to 107, and an LCDR3 according to any one of SEQ ID NOs: 108 to 110, (vi) an LCDR1 according to any one of SEQ ID NOs: 124 to 126, an LCDR2 according to any one of SEQ ID NOs: 127 to 129, and an LCDR3 according to any one of SEQ ID NOs: 130 to 132, (vii) an LCDR1 according to any one of SEQ ID NOs: 146 to 148, an LCDR2 according to any one of SEQ ID NOs: 149 to 150, and an LCDR3 according to any one of SEQ ID NOs: 151 to 152, (viii) an LCDR1 according to any of SEQ ID NOs: 168 to 170, an LCDR2 according to any of SEQ ID NOs: 171 to 173, and an LCDR3 according to any one of SEQ ID NOs: 174 to 176, (ix) an LCDR1 according to any of SEQ ID NOs: 190 to 192, an LCDR2 according to any of SEQ ID NOs: 193 to 195, and an LCDR3 according to any one of SEQ ID NOs: 196 to 198, or (x) an LCDR1 according to any of SEQ ID NOs: 212 to 214, an LCDR2 according to any of SEQ ID NOs: 215 to 217, and an LCDR3 according to any one of SEQ ID NOs: 218 to 220.

[0012] In some embodiments, the chimeric antigen receptor comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), wherein the three HCDRs comprise HCDR1, HCDR2, and HCDR3, and the three LCDRs comprise LCDR1, LCDR2, and LCDR3, and the HCDRs and LCDRs are selected from the group consisting of: (i) an HCDR1 according to any one of SEQ ID NOs: 3 to 5, an HCDR2 according to any one of SEQ ID NOs: 6 to 8, and an HCDR according to any one of SEQ ID NOs: 9 to 11. R3, an LCDR1 according to any one of SEQ ID NOs: 14 to 16, an LCDR2 according to any one of SEQ ID NOs: 17 to 19, an LCDR3 according to any one of SEQ ID NOs: 20 to 22, (ii) an HCDR1 according to any one of SEQ ID NOs: 25 to 27, an HCDR2 according to any one of SEQ ID NOs: 28 to 30, an HCDR3 according to any one of SEQ ID NOs: 31 to 33, an LCDR1 according to any one of SEQ ID NOs: 36 to 38, an LCDR2 according to any one of SEQ ID NOs: 39 to 41, and an LCDR3 according to any one of SEQ ID NOs: 42 to 44. (iii) an HCDR1 according to any one of SEQ ID NOs: 47 to 49, an HCDR2 according to any one of SEQ ID NOs: 50 to 52, an HCDR3 according to any one of SEQ ID NOs: 53 to 55, an LCDR1 according to any one of SEQ ID NOs: 58 to 60, an LCDR2 according to any one of SEQ ID NOs: 61 to 63, and an LCDR3 according to any one of SEQ ID NOs: 64 to 66; (iv) an HCDR1 according to any one of SEQ ID NOs: 69 to 71, an HCDR2 according to any one of SEQ ID NOs: 72 to 74, an LCDR3 according to any one of SEQ ID NOs: 75 to 76, HCDR3 according to any one of SEQ ID NOs: 77, LCDR1 according to any one of SEQ ID NOs: 80 to 82, LCDR2 according to any one of SEQ ID NOs: 83 to 85, LCDR3 according to any one of SEQ ID NOs: 86 to 88, (v) HCDR1 according to any one of SEQ ID NOs: 91 to 93, HCDR2 according to any one of SEQ ID NOs: 94 to 96, HCDR3 according to any one of SEQ ID NOs: 97 to 99, LCDR1 according to any one of SEQ ID NOs: 102 to 104, LCDR2 according to any one of SEQ ID NOs: 105 to 107,(vi) an LCDR3 having any one of SEQ ID NOs: 108 to 110, (vi) an HCDR1 having any one of SEQ ID NOs: 113 to 115, an HCDR2 having any one of SEQ ID NOs: 116 to 118, an HCDR3 having any one of SEQ ID NOs: 119 to 121, an LCDR1 having any one of SEQ ID NOs: 124 to 126, an LCDR2 having any one of SEQ ID NOs: 127 to 129, an LCDR3 having any one of SEQ ID NOs: 130 to 132, (vii) an HCDR1 having any one of SEQ ID NOs: 135 to 137, an HCDR2 having any one of SEQ ID NOs: 138 to 140, and an LCDR3 having any one of SEQ ID NOs: 141 to 143, (viii) an HCDR1 according to any of SEQ ID NOs: 157 to 159, an HCDR2 according to any of SEQ ID NOs: 160 to 162, an HCDR3 according to any one of SEQ ID NOs: 163 to 165, an LCDR1 according to any of SEQ ID NOs: 168 to 170, an LCDR2 according to any of SEQ ID NOs: 171 to 173, and an LCDR3 according to any one of SEQ ID NOs: 174 to 176; (ix) an HCDR1 according to any of SEQ ID NOs: 179 to 181, an LCDR2 according to SEQ ID NO: 18 HCDR2 according to any one of SEQ ID NOs: 182 to 184, HCDR3 according to any one of SEQ ID NOs: 185 to 187, LCDR1 according to any one of SEQ ID NOs: 190 to 192, LCDR2 according to any one of SEQ ID NOs: 193 to 195, LCDR3 according to any one of SEQ ID NOs: 196 to 198, or (x) HCDR1 according to any one of SEQ ID NOs: 201 to 203, HCDR2 according to any one of SEQ ID NOs: 204 to 206, HCDR3 according to any one of SEQ ID NOs: 207 to 209, LCDR1 according to any one of SEQ ID NOs: 212 to 214, LCDR2 according to any one of SEQ ID NOs: 215 to 217, and 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 the three HCDRs and a light chain variable domain comprising the three LCDRs, wherein (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO:1, 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 the three HCDRs and a light chain variable domain comprising the three LCDRs, wherein (i) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 1, and the light chain variable domain is at least 80% identical to SEQ ID NO: 12, (ii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 23, and the light chain variable domain is at least 80% identical to SEQ ID NO: 34, (iii) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 45, and the light chain variable domain is at least 80% identical to SEQ ID NO: 56, (iv) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 67, and the light chain variable domain is at least 80% identical to SEQ ID NO: 78, and (v) the heavy chain variable domain is at least 80% identical to SEQ ID NO: 89, and (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.

[0013] In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs and three LCDRs are comprised in a single polypeptide. In some embodiments comprising three HCDRs and three LCDRs, the three HCDRs are comprised in a first polypeptide and the three LCDRs are comprised in a second polypeptide. In some embodiments, the first 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 is selected from the group consisting of (i) 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, and / or (ii) a binding motif that specifically binds to an antigen selected from the group consisting of HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Rα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B-cell activating factor receptor), and combinations thereof, and / or (ii) a binding motif that specifically binds to an antigen characteristic of B cells, optionally wherein the B cell characteristic antigen is not CD19 or CD20. In some embodiments, the chimeric antigen receptor further comprises an anti-CD19 binding motif.

[0014] In some embodiments, in the chimeric antigen receptor of claim 40, 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 of SEQ ID NOs: 223 to 225, HCDR2 according to any of SEQ ID NOs: 226 to 228, HCDR3 according to any one of SEQ ID NOs: 229 to 231, LCDR1 according to any of SEQ ID NOs: 234 to 236, LCDR2 according to any of SEQ ID NOs: 237 to 239, and LCDR3 according to any one of SEQ ID NOs: 240 to 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 in a single polypeptide.

[0015] 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 together in a single polypeptide. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that is a transmembrane domain of 4-1BB / CD137, a T cell receptor alpha chain, a T cell receptor beta chain, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a T cell receptor zeta chain, or any combination thereof.

[0016] In various embodiments, the present disclosure provides a method for the preparation of a chimeric antigen receptor comprising the steps of: combining a first chimeric antigen receptor of the present disclosure with one or more of the following chimeric antigen receptors: 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, the HER1-HER2 combination, the HER2-HER3 combination, HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Rα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B-cell activating factor receptor), and combinations thereof, and / or a second chimeric antigen receptor comprising a binding motif that specifically binds to an antigen characteristic of B cells, where optionally the B-cell characteristic antigen is not CD19 or CD20. In some embodiments, the second chimeric antigen receptor comprises an anti-CD19 binding motif.

[0017] Additionally, the present disclosure 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 of making engineered cells, comprising transfecting or transducing a cell with a nucleic acid of the present disclosure. In various embodiments, the present disclosure includes a cell that encodes or expresses a chimeric antigen receptor as provided herein, optionally wherein the cell is an immune cell, and optionally wherein the cell is a T cell.

[0018] The present disclosure further provides a method of treating cancer in a subject in need thereof, 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. The present disclosure further provides a method of eliciting an immune response in a subject or immunizing a subject against cancer, 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 some embodiments, the cells are CAR-T cells. In various embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, 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 syndromes, non-Hodgkin's lymphoma (NHL), and thyroid cancer. plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma)), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including dysplasmocyte proliferation; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or Waldenstrom's macroglobulinemia, or a combination thereof. In some embodiments, the cell therapy is allogeneic or autologous cell therapy.

[0019] In at least one aspect, the disclosure includes a chimeric antigen receptor comprising an anti-CD20 binding motif and a CD19 binding motif, wherein the anti-CD20 binding motif comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 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, SEQ ID NO: 199, 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, and SEQ ID NO: 210. In some embodiments, the CD19 binding motif is , SEQ ID NO:221, and SEQ ID NO:232. In some embodiments, the anti-CD20 binding motif comprises an amino acid sequence selected from the group consisting of 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, SEQ ID NO:199, 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, and SEQ ID NO:210, wherein the CD19 binding motif comprises an amino acid sequence selected from the group consisting of SEQ ID NO:221 and SEQ ID NO:232. In some embodiments, the anti-CD20 binding motif and the CD19 binding motif are comprised in a single polypeptide. In some embodiments, the anti-CD20 binding motif and the CD19 binding motif are comprised in different polypeptides.

[0020] In at least one aspect, the disclosure includes a polynucleotide encoding an amino acid sequence selected from the group consisting of 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, SEQ ID NO:199, 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, and SEQ ID NO:210. In at least one aspect, the disclosure includes 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, 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, SEQ ID NO:199, 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, and SEQ ID NO:210. In some embodiments, the composition further comprises a CD19 binding motif. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present disclosure relates to novel polypeptides, including novel antigen-binding molecules, and polynucleotides encoding the polypeptides. Some aspects of the present disclosure relate to polynucleotides encoding chimeric antigen receptors (CARs) comprising at least one of the heavy and light chains (or their CDRs) 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 CARs or TCRs and compositions comprising such polynucleotides. Furthermore, the present disclosure provides engineered cells (e.g., T cells) comprising such polynucleotides and / or transduced with such viral vectors, as well as compositions comprising such engineered cells. The present disclosure also provides compositions (e.g., pharmaceutical compositions) comprising a plurality of engineered T cells. The present disclosure also provides methods for producing such engineered T cells and compositions, as well as uses of such engineered T cells and compositions (e.g., in treating melanoma). The present disclosure also provides methods for inducing immunity against tumors, comprising administering to a subject an effective amount of cells comprising a polynucleotide, vector, or polypeptide of the present disclosure. Other aspects of the present disclosure relate to cells comprising the above-described CARs and the use of the cells in T cell therapy for the treatment of patients suffering from cancer.

[0022] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. While the disclosure is described in conjunction with its detailed description, the above description is intended to be illustrative and not limiting of the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, dictionaries, documents, manuscripts, and scientific literature cited herein are incorporated by reference. Other features and advantages of the present disclosure will become apparent from the following detailed description, including the examples, and from the claims.

[0023] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0024] 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.

[0025] Unless specifically stated or clear from context, the term "or" as used herein is understood to include and encompass both "or" and "and."

[0026] As used herein, the term "and / or" is understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used herein in a phrase 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" used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: 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).

[0027] The terms "for example" and "i.e." as used herein are used by way of example only, without any limitation, and should not be construed as referring only to those items expressly listed herein.

[0028] Terms such as "more than," "at least," and "more than," e.g., "at least one," are used to mean, 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, 110, 111, 112, 113, 114, 115, 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, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the values ​​shown. Also included is any higher number or fraction in between.

[0029] Conversely, the term "less than" includes every value less than the indicated value. For example, "100 nucleotides or less" means 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, etc. , 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. Also included is any smaller number or fraction in between.

[0030] Terms such as "plurality," "at least two," "two or more," "at least a second," and the like are intended to mean, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 7, 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 , 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, and any higher number or fraction therebetween is also included.

[0031] Throughout this specification, the words "comprising" or variations such as "comprises" or "comprising" mean that the elements indicated, integers or integers, are all elements that are part of the invention. It is understood that any reference herein implies the inclusion of an element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps. Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0032] Unless specifically stated or clear from the context, the term "about" as used herein refers to a value or composition that falls within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, and 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 "comprising essentially of" are used in the art to mean any value that falls within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, and will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. "About" or "essentially comprising" can mean within a range of one or more standard deviations per run. "About" or "essentially comprising" can mean within a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to include a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the indicated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to 10 times or up to 5 times a value. When specific values ​​or compositions are provided in this disclosure, unless otherwise indicated, "about" or "essentially comprising" is intended to mean within an acceptable range of error for that particular value or composition.

[0033] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated.

[0034] Units, prefixes and symbols used herein are presented using their accepted form of the International System of Units (SI). Numerical ranges are expressed as the sum of the units within the range. Contains the numerical values ​​that define the range.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, e.g., Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2 nd ed., (2001), CRC Press, "The Dictionary of Cell & Molecular Biology", 5 th ed., (2013), Academic Press, and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et al. eds., 2 nd ed., (2006), Oxford University Press, will provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0036] "Administering" can be carried out in a variety of ways known to those skilled in the art. "Parenteral administration" refers to the physical introduction of an agent into a subject using any of a variety of delivery systems. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, sub-articular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulations are administered by a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical, and administration may be, for example, single, multiple, and / or over one or more extended periods of time.

[0037] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, antibodies may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, inter-connected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, namely, 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, namely, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). VH and VL each contain three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: 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 regions of Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD), which associate with each other in what is commonly referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to each other by a single disulfide bond, with two other disulfide bonds connecting the heavy chain hind chains. The dimers are connected to each other, thus forming tetramers. Naturally produced antibodies are also glycosylated, for example on the CH2 domain.

[0038] The term "human antibody" is intended to include antibodies having variable and constant domain sequences prepared, assembled, or derived from human immunoglobulin sequences or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) can be considered "human" even if their amino acid sequences contain residues or elements not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "humanized" is intended to include antibodies having variable domains derived from the variable domains of a non-human species (e.g., mouse) and having sequences modified to more closely resemble human germline-encoded sequences. In some embodiments, a "humanized" antibody comprises one or more framework domains that have substantially the amino acid sequence of a human framework domain and one or more complementarity-determining regions that have 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 at least a portion of a human immunoglobulin constant domain. In some embodiments, the humanized antibody comprises a C H 1 region, hinge region, C H 2 areas, C H 3 regions, and optionally C H It may contain four regions.

[0039] Antibodies 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 molecules 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 (referred to herein as "antibodies"). Examples of antibodies include polyclonal antibodies (sometimes referred to herein as "antibody mimetics"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, antibodies as described herein refer to polyclonal antibody populations. Antibodies can 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 IgNARs or fragments thereof), camelized antibodies, single chain diabodies or tandem diabodies (TandAb™), Anticalin™, Nanobody™, minibody, BiTE™, ankyrin repeat protein or DARPIN™, Avimer™, DART, TCR-like antibodies, Adnectin™, Affilin™, Trans-body™, Affibody™, TrimerX™, MicroProtein, Fynomer™, Centyrin™, and KALBITOR™.

[0040] Immunoglobulins can be derived from any of the commonly known isotypes, 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 genes. The term "antibody" includes, by way of example, Abs of both natural and non-natural origin, monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; and total antibody. "antibody" includes non-human Abs; synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and unless otherwise indicated, the term "antibody" also includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain Abs.

[0041] An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule comprising the antigen-binding portion of an antibody (e.g., CDR), which molecule is derived from that antibody. An antigen-binding molecule may comprise antigen-complementarity 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 antibodies containing a peptide-binding domain) are also known. A CD40 antibody (CD40A) is another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or to a viral or bacterial antigen. In certain embodiments, the antigen-binding molecule binds to BCMA, CLL-1, or FLT3. In certain embodiments, the antigen-binding molecule binds to CD19, CD20, or both. In further embodiments, the antigen-binding molecule is an antibody fragment that specifically binds to an antigen, comprising one or more complementarity-determining regions (CDRs) thereof. In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule comprises or consists of an avimer.

[0042] In some cases, the CDRs are substantially identical in sequence to a CDR found in a reference antibody (e.g., an antibody of the present disclosure) and / or a CDR provided in this disclosure. In some embodiments, the CDRs are substantially identical to a reference CDR (e.g., a CDR provided in this disclosure) in that they are either identical in sequence or contain between 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions compared to the reference CDR. In some embodiments, the CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) sequence identity with the reference CDRs. In some embodiments, the CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, a CDR is substantially identical to a reference CDR in that one amino acid within the CDR has been deleted, added, or substituted relative to the reference CDR, but the CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, a CDR is substantially identical to a reference CDR in that two, three, four, or five (e.g., two to five) amino acids within the CDR have been deleted, added, or substituted relative to the reference CDR, but 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.

[0043] Antigen-binding fragments can be produced by any means. For example, in some embodiments, antigen-binding fragments can be produced enzymatically or chemically by fragmentation of an intact antibody. In some embodiments, antigen-binding fragments can be produced recombinantly (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, antigen-binding fragments can be wholly or partially synthetically produced. In some embodiments, antigen-binding fragments are at least about 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 130 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, 180 amino acids, 190 amino acids or more in length, and in some embodiments, at least The length may be approximately 200 amino acids (for example, 50 to 100 amino acids, 50 to 150 amino acids, 50 to 200 amino acids, or 100 to 200 amino acids).

[0044] The terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically about the amino-terminal 110-120 amino acids in a mature heavy chain and about 90-115 amino acids in a mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody with an antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0045] 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.

[0046] 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.

[0047] Many definitions of CDRs are in common use: 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 an analysis of available complex crystal structures.

[0048] TIFF2025169340000001.tif73170

[0049] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody, or an antigen-binding molecule thereof. In certain embodiments, the CDRs of an antibody can be identified according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391, and Kabat EA et al., (1991) Sequences of Proteins of Immunol (See, for example, Journal of Biological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242.) Uses the Kabat numbering system. Thus, the CDRs in an antibody heavy chain molecule are typically located at amino acids 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3), which may optionally include one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acids 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). In specific embodiments, the CDRs of the antibodies described herein were determined according to the Kabat numbering scheme.

[0050] In certain embodiments, the CDRs of an antibody may be identified according to the Chothia numbering scheme, which refers to the location of the structural loops of an immunoglobulin (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948, Chothia C et al., (1992) J Mol Biol 227: 799-817, Tramontano A et al., (1990) J Mol Biol 215(1): 175-82, and U.S. Patent No. 7,709,226. Typically, using the Kabat numbering convention, the CDR-H1 loop of Chothia is located at amino acids 26-32, 33, or 34 of the heavy chain, the CDR-H2 loop of Chothia is located at amino acids 52-56 of the heavy chain, and the CDR-H3 loop of Chothia is located at amino acids 95-102 of the heavy chain, whereas the CDR-L1 loop of Chothia is located at amino acids 24-34 of the light chain, the CDR-L2 loop of Chothia is located at amino acids 50-56 of the light chain, and the CDR-L3 loop of Chothia is located at amino acids 89-97 of the light chain. The end of the Chothia CDR-HI loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B, i.e., if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). In specific embodiments, the CDRs of the antibodies described herein were identified according to the Chothia numbering scheme.

[0051] The terms "constant region" and "constant domain" are used interchangeably and have a common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that 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 compared to the immunoglobulin variable domain.

[0052] The term "heavy chain", when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4, based on the amino acid sequence of the constant domain.

[0053] The term "light chain," when used in reference to an antibody, can refer to any of the different types, e.g., 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.

[0054] "Binding affinity" generally refers to the strength of the total 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, "binding affinity" as used herein refers to the strength of the total non-covalent interactions between a member of a binding pair (e.g., an antigen). The affinity of a molecule X for its partner Y is generally expressed as the dissociation constant (K D Affinity can be expressed by, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art. D is k off / k on It is calculated from the quotient of K A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k off can be determined by techniques known to those skilled in the art, such as BIACORE™ or KinExA.

[0055] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within the CDR(s) or framework region(s) of an antibody or antigen-binding molecule thereof can be substituted with an amino acid residue having a similar side chain. In general, two sequences are generally considered to be "substantially similar" if they contain conservative amino acid substitutions at corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Replacing one amino acid with another of the same type may be considered a conservative substitution. Exemplary amino acid categorizations are summarized in Tables 2 and 3 below.

[0056] TIFF2025169340000002.tif114170

[0057] TIFF2025169340000003.tif32170

[0058] The term "heterologous" means from any source other than a sequence of natural origin. For example, a heterologous sequence included as part of a costimulatory protein having the amino acid sequence of SEQ ID NO: 232, e.g., the corresponding human costimulatory protein, contains amino acids that do not naturally occur as, i.e., do not align with, the wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the wild-type human costimulatory protein coding sequence.

[0059] "Epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (a linear or contiguous epitope), or an epitope can be, for example, derived from two or more non-contiguous regions of a polypeptide(s) together (a conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be identified by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography studies, crystallization is carried out according to methods 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 well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., U.S. Pat. Appl. Pub. No. 2005 / 0100122). The mutagenesis mapping can be refined using computer software such as RT-PCR (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. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394, and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0060] An antigen-binding molecule, antibody, or antigen-binding molecule thereof "cross-competes" with a reference antibody or its antigen-binding molecule if the interaction of the antigen with the first binding molecule, antibody, or antigen-binding molecule thereof blocks, limits, inhibits, or reduces the ability of the reference binding molecule, reference antibody, or its antigen-binding molecule to interact with the antigen. Cross-competition can be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or cross-competition can be partial, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind to the antigen. In certain embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds a different epitope than the reference antigen-binding molecule. Many types of competitive binding assays For example, solid-phase direct or indirect radioimmunoassays (RIA); solid-phase direct or indirect enzyme immunoassays (EIA); sandwich competitive assays (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 assays, solid-phase direct label sandwich assays (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I125 label (Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82) can be used to determine whether one antigen-binding molecule competes with another.

[0061] The term "binding" generally refers to a non-covalent association between two or more entities. Direct binding includes physical contact between the entities or moieties. "Indirect" binding includes physical interaction through physical contact with one or more intervening 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 more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system such as a cell).

[0062] The terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are analogous terms with respect to antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex), as such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassay, BIACORE™, KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho), or other assays known in the art. In specific embodiments, a molecule that specifically binds to an antigen has a K A Compared to K by at least 2 log, 2.5 log, 3 log, or 4 log A The binding may include preferential binding of the binding motif, antibody, or antigen-binding system to the target of the binding motif, antibody, or antigen-binding system compared to binding of the binding motif, antibody, or antigen-binding system to an entity that is not the target (i.e., a non-target). In some embodiments, a binding motif, antibody, or antigen-binding system selectively binds to a target when the binding between the binding motif, antibody, or antigen-binding system and the target is more than 2-fold, more than 5-fold, more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold, or more than 100-fold greater than the binding of the binding motif, antibody, or antigen-binding system to the non-target. In some embodiments, the binding affinity is about 10 -5 Less than M, about 10 -6 Less than M, about 10-7 Less than M, about 10 -8 Less than M or about 10 -9 If M is less than M, then the binding motif, antibody, or antigen-binding system selectively binds to the target.

[0063] In another embodiment, the molecules that specifically bind to an antigen are about 1 x 10 -7 Dissociation constant of M (K d In some embodiments, the antigen-binding molecule binds at K d is about 1×10 -9 M ~ approx. 5×10 -9 In some embodiments, the antigen-binding molecule specifically binds an antigen with a "high affinity" when K d is about 1×10 -10 M ~ approx. 5×10 -10 In one embodiment, the antigen-binding molecule specifically binds the antigen with "very high affinity" when the affinity is 10 -9 K of M d In one embodiment, the off-rate is about 1×10 -5 In other embodiments, the antigen-binding molecule is less than about 1 x 10 -7 M ~ approx. 1×10 -13 K of M d In yet another embodiment, the antigen-binding molecule binds human BCMA at about 1 x 10 -10 M ~ approx. 5×10 -10 K of M d In some embodiments, the antigen binding molecule binds human CD19, CD20, or both at about 1 x 10 -7 M ~ approx. 1×10 -13 K of M d In yet another embodiment, the antigen-binding molecule binds to human CD19, CD20, or both at about 1 x 10 -10 M ~ approx. 5×10 -10 K of M d Join with .

[0064] In specific embodiments, provided herein are antibodies or antigen-binding molecules thereof that bind to a target human antigen, e.g., human BCMA or human CLL-1, with higher affinity than to a target antigen of another species, e.g., non-human BCMA or non-human CLL-1. In some embodiments, provided herein are antibodies or antigen-binding molecules thereof that bind to human CD19, human CD20, or both, with higher affinity than to one or both target antigens of another species, e.g., non-human CD19, non-human CD20, or both. In certain embodiments, the affinity is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% higher than to a target antigen of another species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. Presented herein are antibodies or antigen-binding molecules thereof that bind to a target human antigen, such as human BCMA or human CLL-1, with 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% greater affinity. In certain embodiments, presented 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%, or 70% greater affinity than to one or both target antigens of another species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In specific embodiments, an antibody or antigen-binding molecule thereof described herein that binds to a target human antigen binds to a target antigen of another species with less than 10%, 15%, or 20% of the binding of the antibody or antigen-binding molecule thereof to the human antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

[0065] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to contain a binding motif and a means to activate an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells engineered to express a chimeric antigen receptor may be referred to as CAR T cells. "Extracellular domain" (i.e., "ECD") refers to the portion of a polypeptide that, when present in the cell membrane, is understood to be present outside the cell membrane, i.e., in the extracellular space.

[0066] "Antigen" refers to any molecule capable of eliciting an immune response or being bound by an antibody or antigen-binding molecule. The immune response can involve either antibody production or the activation of specific immunologically competent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be specific to a particular tissue, such as cancer cells, or can be widely expressed. Additionally, fragments of larger molecules can act as antigens. In one embodiment, the antigen is a tumor antigen. In a particular embodiment, the antigen is all or a fragment of CD19 or CD20. A "target" is any molecule that is bound by a binding motif, antigen-binding system, or binding agent, e.g., an antibody. In some embodiments, the target is an antigen or epitope of the present disclosure.

[0067] The term "neutralize" 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 by indirect means (such as by altering the structure or energy of the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the bound protein from performing a biological function.

[0068] The term "autologous" refers to any material derived from the same individual that is to be later reintroduced. For example, the engineered autologous cell therapy (eACT™) described herein involves the collection of lymphocytes from a patient, which are then engineered, for example, to express a CAR construct and then administered back to the same patient.

[0069] The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species (eg, allogeneic T cell transplantation).

[0070] The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell by 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 papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0071] "Transformation" refers to any process by which exogenous DNA is introduced into a host cell. Transformation can be performed using a variety of methods under natural or artificial conditions. Transformation can be achieved using any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. In some embodiments, several transformation methodologies are selected based on the host cell to be transformed and / or the nucleic acid to be inserted. Transformation methods may include, but are not limited to, viral infection, electroporation, and lipofection. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA can replicate either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cell can express the introduced nucleic acid.

[0072] The term "vector" refers to a recipient nucleic acid molecule that contains or has been modified to include a designated nucleic acid sequence. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Furthermore, certain vectors contain sequences that direct the expression of inserted genes to which they are operably linked. Such vectors may be referred to herein as "expression vectors." See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Standard techniques can be used to manipulate vectors, such as those found in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference for all purposes.

[0073] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous 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 lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods of the present disclosure are used to treat cancers of the immune system, including, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), and other cancers of the immune system, including, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, 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), and other cancers of the immune system, including, for example ... B cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue cancer 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 of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma The present invention may be used to reduce the size of tumors derived from tumors derived from thyroid cancer, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. In one particular embodiment, the cancer is multiple myeloma. Certain cancers may be responsive to chemotherapy or radiation therapy, while others may be refractory. Refractory cancer refers to cancer that is not amenable to surgical treatment; the cancer is either initially unresponsive to chemotherapy or radiation therapy, or the cancer becomes unresponsive over time. Additionally, cancers include diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma after two or more lines of systemic therapy, high-grade B-cell lymphoma, and relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including DLBCL arising from follicular lymphoma.

[0074] As used herein, "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors. Anti-tumor effect can also refer to the prevention of tumor development, e.g., vaccination.

[0075] As used herein, "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where the cytokine interacts with a second cell and mediates a response in the second cell. Cytokines can be endogenously expressed by a cell or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines include homeostatic cytokines, chemokines, pro-inflammatory cytokines, and inflammatory cytokines. These cytokines may include cytokines, effector and acute phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, may promote the survival and proliferation of immune cells, while proinflammatory cytokines may promote the inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, 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 acute phase proteins 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).

[0076] A "chemokine" is a type of cytokine that mediates cell chemotaxis or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0077] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, e.g., an engineered CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability attributable to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled physician, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0078] The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that constitutes a major component of the innate immune system. NK cells reject tumor and virus-infected cells. NK cells act by the process of apoptosis, or programmed cell death. NK cells are named "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (antibody-independent). Their T cell receptor (TCR) differentiates them from other types of lymphocytes. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells (i.e., stem cell memory T cells such as naive cells), and SCM The cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells; (ii) central memory T CM cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory T EM There are various types of T cells, including T cells (which do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells. On the other hand, B cells play the most important role in humoral immunity (antibody-mediated immunity). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and, after activation by antigen interaction, become memory B cells. In mammals, immature B cells are formed in the bone marrow, from which they are named.

[0079] "Linker" (L) or "linker domain" or "linker region" refers to an oligopeptide or polypeptide region, approximately 1 amino acid to 100 amino acids in length, that links together any of the domains / regions of the CAR of the invention. Linkers can be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely in relation to one another. The linker can move in a circular fashion. Longer linkers can be used when it is desirable to prevent two adjacent domains from sterically interfering with each other. The linker can 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 porcine teschovirus (P2A), a CHYSEL sequence of a virus (T2A), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence is Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A) -Pro (2B) The linker may comprise a motif (SEQ ID NO: 314) resulting in a cleavage between the glycine at 2A and the proline at 2B. Other linkers will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the invention. For example, in some instances, a linker may be used to connect or link different antigen binding systems, such as two CARs of a bicistronic CAR. The linker may be part of a multi-element agent that connects different elements to one another. For example, a polypeptide may comprise two or more functional or structural domains, with a stretch of amino acids between such domains linking them together. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, where S1 and S2 represent two domains, which may be the same or different, that are joined together by the linker. The linker can connect or link together any of the domains / regions of a CAR of the present disclosure. In some embodiments, the polypeptide linker comprises at least 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, 55 amino acids, 60 amino acids, 65 amino acids, 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids. , 90, 95, 100 or more amino acids in length (e.g., 1 to 10 amino acids, 1 to 20 amino acids, 1 to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, 1 to 100 amino acids, 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, or 10 to 100 amino acids in length). In some embodiments, the linker is characterized in that it does not adopt a rigid three-dimensional structure, but instead tends to impart flexibility to the polypeptide.

[0080] "Single-chain variable fragment," "single-chain antibody variable fragment," or "scFv" antibody refers to a form of antibody that contains only the variable regions of the heavy and light chains connected by a linker peptide.

[0081] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of a cell, including, but not limited to, the deletion of coding or non-coding regions, or portions thereof, or the insertion of coding regions, or portions thereof. In some embodiments, the cells being modified are lymphocytes, e.g., T cells, which can be obtained from either a patient or a donor. T cells can be engineered to express a foreign construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the genome of the cell. Engineering generally involves manipulation by the hand of man. For example, a polynucleotide can be engineered by the hand of man so that two or more sequences that are not naturally linked or connected together in that order are directly linked or connected to each other in the engineered polynucleotide. In the context of manipulating cells with molecular biology techniques, a cell or organism is considered "engineered" when it has been manipulated in such a way that its genetic information is altered (e.g., when new genetic material not previously present is introduced, e.g., by transformation, somatic cell hybridization, transfection, transduction, or other mechanisms, or when previously present genetic material is altered or removed, e.g., by substitution or deletion mutations or other protocols). In some embodiments, the binding agent is an engineered lymphocyte, e.g., a T cell, which can be obtained from a patient or a donor. Engineered cells can be engineered to express a foreign construct, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR), that is integrated into the genome of the cell. The result of an engineered polynucleotide or binding agent is generally referred to as "engineered," even if the actual manipulation was performed on a previous entity. In some embodiments, "engineered" refers to an entity being designed and produced. The term "designed" refers to (i) an agent whose structure is selected or chosen by human intervention, (ii) an agent that is produced by a process requiring human intervention, and / or (iii) an agent that differs from natural products and other known agents. "T cell receptor," or "TCR," refers to the antigen-recognition molecule present on the surface of T cells. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to result in a wide variety of TCR proteins.

[0082] "Immune response" refers to the actions 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 (including Abs, cytokines, and complement) produced by any of these cells or the liver that result in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0083] The term "immunotherapy" refers to the treatment of a subject afflicted with a disease or at risk of developing a disease or suffering a recurrence of a disease by methods that involve inducing, enhancing, suppressing, or modifying the immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy includes adoptive T cell therapy, tumor These treatments may include T-cell infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T-cell transplantation. However, one skilled in the art will recognize that the conditioning methods disclosed herein may enhance the efficacy of any transplanted T-cell therapy. Examples of T-cell therapies are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and WO 2008 / 081035.

[0084] T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be isolated from a subject using any of a number of techniques known to those skilled in the art, such as FICOLL™ isolation and / or apheresis. They may also be obtained from a unit of blood collected from the body. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0085] "Engineered Cell Transfer," which can be abbreviated as "eACT™" and is also known as adoptive cell transfer The term "autologous cell therapy" refers to the process of collecting a patient's own T cells and then genetically modifying 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) with specificity for a particular tumor antigen linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. The costimulatory domain can be derived from, for example, a naturally occurring costimulatory domain having the amino acid sequence of SEQ ID NO: 232, or a variant thereof, such as a variant with 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 costimulatory domains. CAR scFvs can be designed to target CD19, 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, CARs are engineered to express a costimulatory domain as a separate polypeptide chain. Examples of CAR T-cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties. "Adoptive cell therapy," or "ACT," involves the transfer of immune cells with anti-tumor activity into a subject, e.g., a cancer patient. In some embodiments, ACT is a therapeutic approach involving the use of lymphocytes (e.g., engineered lymphocytes) with anti-tumor activity.

[0086] A "patient" includes any human afflicted with cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0087] The term "in vitro" refers to events that take place in an artificial environment, such as a test tube, reaction vessel, cell culture, etc., rather than within a multicellular organism. The term "in vitro cell" refers to any cell that is cultured ex vivo. In particular, in vitro cells can include T cells. The term "in vivo" refers to events that take place within a multicellular organism, such as a human or non-human animal.

[0088] An "antigen-specific targeting region" (ASTR) refers to a region of a CAR that targets a specific antigen. The CAR of the present invention comprises at least two targeting regions that target at least two different antigens. In one embodiment, the CAR comprises three or more targeting regions that target at least three or more different antigens. The targeting regions on the CAR are extracellular. In some embodiments, the antigen-specific targeting region comprises an antibody or its functional equivalent or a fragment or derivative thereof, with each targeting region targeting 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 diabody, each of which is specific for a target antigen. However, many options exist, such as linked cytokines (leading to recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains derived from naturally occurring receptors, soluble protein / peptide ligands for receptors (e.g., on tumor cells), peptides, and vaccines that stimulate an immune response, each of which may be used in various embodiments of the present invention. Indeed, as will be appreciated by those skilled in the art, almost any molecule that binds with high affinity to a given antigen can be used as an antigen-specific targeting moiety.

[0089] "Antigen-presenting cells" or "APCs" refer to cells that process and present antigens 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 costimulation.

[0090] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the terms also refer to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0091] As used herein, "stimulation" refers to a primary response induced by binding of a stimulatory molecule with its cognate ligand, where the binding mediates a signal transduction event. A "stimulatory molecule" refers to a molecule on a T cell, e.g., the T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, T cell activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies (e.g., OKT3), peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0092] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, such as, but not limited to, T cell proliferation and / or upregulation or downregulation of key molecules.

[0093] As used herein, "costimulatory ligand" includes molecules on antigen-presenting cells that specifically bind cognate costimulatory molecules on T cells. Binding of a costimulatory ligand provides signals that mediate T cell responses, including, but not limited to, T cell proliferation, activation, differentiation, etc. Costimulatory ligands induce signals in addition to the primary signal provided by stimulatory molecules, for example, by binding of the T cell receptor (TCR) / CD3 complex with a peptide-loaded major histocompatibility complex (MHC) molecule. Examples of costimulatory ligands include, but are not limited to, 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT, and the like. Examples of costimulatory ligands include 4, immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin β receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, and programmed cell death (PD) L1. Examples of costimulatory ligands include, but are not limited to, 4-1BB, B7-H3, a ligand that specifically binds to CD2, CD27, CD28, CD30, CD40, CD7, ICOS, and CD83, and antibodies that specifically bind to costimulatory molecules present on T cells, such as 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).

[0094] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. As a costimulatory molecule, without limitation, a "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.Costimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3(α;β;δ;ε;γ;zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, C D80, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Igα (CD79a), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation 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.

[0095] The terms "reducing" and "decreasing" are used interchangeably herein to refer to any change that is less than what is originally intended. "Reducing" and "reducing" are relative terms that require a comparison of a before and after measurement. "Reducing" and "reducing" include a complete absence.

[0096] The terms "improve," "increase," "inhibit," and "reduce" refer to values ​​relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may include measurements in a particular system (e.g., a single individual) lacking the presence of the agent or treatment (e.g., before and / or after) but under otherwise equivalent conditions, or in the presence of an appropriate equivalent reference agent. In some embodiments, a suitable reference measurement may include measurements in an equivalent system known or expected to respond equivalently in the presence of the relevant agent or treatment.

[0097] "Treatment" of a subject or "treating" a subject " refers to any type of treatment or process performed on a subject, or the administration of an active ingredient to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, delaying, or preventing the onset, progression, manifestation, severity, or recurrence of symptoms, complications, or conditions, or biochemical markers associated with a disease. In one embodiment, "treatment" or "treat" includes partial remission. In another embodiment, "treatment" or "treat" includes complete remission. In some embodiments, treatment includes providing a subject who is free of symptoms of the associated disease, disorder, and / or condition, and In some embodiments, the treatment may be treatment of a subject who exhibits only early signs of a disease, disorder, and / or condition. In some embodiments, the treatment may be treatment of a subject who exhibits one or more established signs of an associated disease, disorder, and / or condition. In some embodiments, the treatment may be treatment of a subject who has been diagnosed with an associated disease, disorder, and / or condition. In some embodiments, the treatment may be treatment of a subject known to have one or more susceptibility factors statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0098] The term "agent" can refer to any class of molecule or entity, including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, cell, or organism (e.g., a fraction or extract thereof) or component thereof, or to a plurality of molecules or entities, any of which is, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, cell, or organism (e.g., a fraction or extract thereof) or component thereof. In some embodiments, an agent may be utilized in isolated or pure form. In some embodiments, an agent may be utilized in crude or impure form. In some embodiments, an agent may be provided as a population, collection, or library, for example, that can be screened to identify or characterize members present therein.

[0099] Two events or entities are "associated" with one another if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, an entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a disease, disorder, or condition if its presence, level, and / or form correlates (e.g., across a relevant population) with the incidence of, and / or susceptibility to, the disease, disorder, or condition. For example, two or more entities are physically "associated" with one another when they interact, either directly or indirectly, such that they come into and / or remain in physical proximity to one another (e.g., bound). In additional examples, two or more entities that are physically associated with one another may be covalently linked or connected to one another or non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic forces, and combinations thereof.

[0100] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for calculating the percent identity between two given polypeptide sequences are known. Calculating the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences can be performed using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100%) identical.

[0101] To calculate percent identity, the sequences to be compared are typically aligned in a way that gives the greatest match between the sequences. One 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, University of Wisconsin, Madison, Wisconsin, Genetics Computer Group). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. The sequences are aligned for the best match of their respective amino acids or nucleotides (the "matched span" as determined by the algorithm). Also, in certain embodiments, the algorithms use standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix). Other algorithms can be used for comparison of amino acid or nucleic acid sequences, including algorithms available in commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences.Exemplary such programs are 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 programs described above generally provide an indication of the degree of similarity. In some embodiments, two sequences are considered substantially similar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100%) of their corresponding residues are similar and / or identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the entire sequence. In some embodiments, the relevant 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 that have significant sequence similarity may be homologous to each other.

[0102] "Combination therapy" refers to a situation in which a subject receives two or more therapeutic regimens (e.g., two or more therapeutic moieties) simultaneously. 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 a first regimen are administered before administration of any dose of a second regimen). In some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy refers to the administration of one or more agents or modalities to a subject to whom other agent(s) or modality(s) are being administered. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more agents or active portions thereof may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalently linked entity).

[0103] "Corresponding to" may 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) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, for simplicity, residues in a polypeptide may be designated using a canonical numbering system based on the reference related polypeptide, so that, for example, an amino acid "corresponding to" a residue at position 100 need not actually be the 100th amino acid in the amino acid chain, as long as it corresponds to the residue at position 100 in the reference polypeptide. For example, various sequence alignment strategies can be used to identify "corresponding" residues in polypeptides and / or nucleic acids according to the present disclosure, including software programs such as BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE.

[0104] The term "domain" refers to a portion of an entity. In some embodiments, a "domain" refers to a structural and / or functional characteristic of an entity, such that, for example, when a domain is physically separated from the remainder of its parent entity, it substantially or completely retains the structural and / or functional characteristic. In some embodiments, a domain can comprise a portion of an entity that, when separated from its (parent) entity and linked or connected to a different (recipient) entity, substantially retains and / or confers to the recipient entity one or more structural and / or functional characteristics, e.g., characteristic of the parent entity. In some embodiments, a domain is a portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide. In some such embodiments, a domain is characterized by structural elements (e.g., amino acid sequence or sequence motif, α-helical character, β-sheet character, coiled-coil character, random coil character, etc.) and / or functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0105] The term "dosage form" may be used to refer to a physically discrete unit of an active agent (e.g., an antigen-binding system or an antibody) for administration to a subject. Generally, each such unit contains a predetermined amount of the active agent. In some embodiments, such amount is a unit dose (or a whole fraction thereof) appropriate for administration according to a dosing regimen recognized to correlate with a desired or beneficial outcome when administered to a relevant population. The total amount of therapeutic composition or therapeutic agent to be administered to a subject is determined by one or more physicians and may involve administration of more than one dosage form.

[0106] The term "dosing regimen" can be used to refer to a series of one or more unit doses administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can include one or more administrations. In some embodiments, a dosing regimen includes multiple administrations that are separated in time from other administrations. In some embodiments, a dosing regimen includes multiple administrations, with successive administrations separated from each other by periods of equal length. In some embodiments, the dosing regimen comprises multiple administrations, with successive administrations separated from each other by at least two different lengths of time. In some embodiments, all administrations within the dosing regimen are administrations of the same unit dosage. In some embodiments, different administrations within the dosing regimen are administrations of different amounts. In some embodiments, the dosing regimen comprises a first administration at a first dosage amount, followed by one or more additional administrations at a second dosage amount different from the first dosage amount. In some embodiments, the dosing regimen is periodically adjusted to achieve a desired or beneficial outcome.

[0107] "Effector function" refers to the biological outcome of the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated cytophagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector functions can be antigen binding-dependent, antigen binding-independent, or both. 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 Fc receptor (FcR)-bearing effector cells that recognize and kill antibody-coated target cells (e.g., cells expressing the antigen to which the antibody binds on their surface). Effector cells that mediate ADCC can include immune cells, including, but not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0108] "Effector cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, effector cells may include, but are not limited to, one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, and B lymphocytes. Effector cells may be from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0109] The term "excipient" refers to an agent that can be included in a composition to, for example, impart or contribute to a 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, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc.

[0110] A "fragment" or "portion" of a material or entity described herein has a structure that includes, for example, a discrete portion of a whole physical or abstract entity. In some embodiments, the fragment lacks one or more portions found in the whole. In some embodiments, the fragment consists of or comprises a characteristic structural element, domain, or portion found in the whole. In some embodiments, a polymer fragment is 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, or more of the monomer units (e.g., residues) found in the whole polymer. , 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. In some embodiments, the polymer fragments comprise at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the monomer units (e.g., residues) found in the entire polymer. , 96%, 97%, 98%, 99% or more (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100%). In some embodiments, the entire material or entity may be referred to as the "parent" of the fragment.

[0111] The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. Generally, a polypeptide comprising at least two such segments is considered to be a fusion polypeptide if the two segments (1) are not naturally contained in the same peptide, and / or (2) have not previously been linked or joined to each other in a single polypeptide, and / or (3) are moieties that have been linked or joined to each other by the act of hand of man.

[0112] The term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or to a polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0113] The term "isolated" refers to a substance (1) that has been separated from at least some components with which it was previously associated or that would otherwise be associated, and / or a substance (2) that is present in a composition that includes limited or defined amounts or concentrations of one or more known or unknown contaminants. In some embodiments, an isolated substance is free from other non-substance components with which it was previously associated, e.g., components with which it was previously associated or that would otherwise be associated. The substance may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100%) of other components or contaminants with which it becomes associated. In certain cases, a substance is isolated when it is present in a composition containing limited or reduced amounts or concentrations of the same or similar types of molecules. For example, in certain cases, a nucleic acid, DNA, or RNA substance is isolated when it is present in a composition containing limited or reduced amounts or concentrations of nucleic acid, DNA, or RNA molecules distinct from the substance. For example, in certain cases, a polypeptide substance is isolated when it is present in a composition containing limited or reduced amounts or concentrations of polypeptide molecules distinct from the substance. In certain embodiments, the amount can be, for example, an amount measured relative to the amount of desired substance present in the composition. In certain embodiments, a limited amount can be an amount that is 100% or less of the amount of substance in the composition, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or less (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100%) of the amount of substance in the composition. In certain instances, a composition is pure or substantially pure with respect to a selected substance. In some embodiments, an 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 greater 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 contaminants. In some embodiments, a substance may be considered "isolated" or even "pure" even 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 percent isolation or purity of a substance is calculated exclusive of such carriers or excipients.

[0114] "Nucleic acid" refers to any polymeric chain of nucleotides. Nucleic acids include DNA, RNA, or or combinations thereof. In some embodiments, the nucleic acid comprises one or more naturally occurring nucleic acid residues. In some embodiments, the nucleic acid is comprised of 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 by polymerization based on a complementary template (in vivo or in vitro), renaturation in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250 residues in length. , 275 residues, 300 residues, 325 residues, 350 residues, 375 residues, 400 residues, 425 residues, 450 residues, 475 residues, 500 residues, 600 residues, 700 residues, 800 residues, 900 residues, 1000 residues, 1500 residues, 2000 residues, 2500 residues, 3000 residues, 3500 residues, 4000 residues, 4500 residues, 5000 residues or more (e.g., 20 to 100 residues, 20 to 500 residues, 20 to 1000 residues, 20 to 2000 residues, or 20 to 5000 residues or more). 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, a nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or at least one element that is the complement of a sequence that encodes a polypeptide.

[0115] "Operably linked" refers to a juxtaposition wherein the described components 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 such that expression and / or activity of the functional element is achieved under conditions compatible with the control element.

[0116] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to the recipient, or any harmful effects are outweighed by the benefit to the recipient. With respect to carriers, diluents, or excipients used to formulate the compositions 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 any harmful effects must be outweighed by the benefit to the recipient. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting an agent from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or any harmful effects must be outweighed by the benefit to the recipient. Some examples of materials which may act 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, powdered tragacanth, 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 glycerin, 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, ethyl alcohol, pH buffers, polyesters, polycarbonates and / or polyanhydrides, and and other non-toxic, compatible substances used in pharmaceutical formulations.

[0117] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, the pharmaceutical composition may be formulated for administration in a solid or liquid form, including, but not limited to, oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., tablets intended for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes applied to the tongue, parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained release formulation, topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity, vaginal or rectal administration, e.g., as a pessary, cream, or foam, sublingual, ocular, transdermal, or nasal, pulmonary, and other mucosal surface adapted forms.

[0118] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, 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 histological reference or control, optionally embedded in a tangible medium. Generally, a reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. There is sufficient similarity to justify reliance on and / or comparison to the selected reference or control.

[0119] "Regulatory T cells" ("Treg," "Treg cells," or "Tregs") refer to a lineage of CD4+ T lymphocytes involved in controlling certain immune activities, such as responses to autoimmunity, allergies, and infections. Regulatory T cells can modulate the activity of T cell populations and can also affect certain innate immune system cell types. Tregs can be identified by expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically comprise approximately 5%-10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells) can represent as much as 20%-30% of the total CD4+ T lymphocyte population.

[0120] The term "sample" generally refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may include a cell or organism, such as a cell population, a tissue, or an animal (e.g., a human). In some embodiments, the source of interest includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, milk, cerebrospinal fluid, earwax, chyle, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, biological fluids may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and / or transcellular fluid. In some embodiments, biological fluids may include plant exudates. In some embodiments, biological tissue or biological samples may be obtained by, for example, aspiration, biopsy (e.g., fine needle biopsy or tissue biopsy), swab (e.g., oral swab, intranasal In some embodiments, a biological sample may be obtained by swabbing, skin swabbing, or vaginal swabbing), scraping, surgery, lavage, or washing (e.g., bronchoalveolar lavage, ductal lavage, nasal wash, ocular wash, oral wash, uterine wash, vaginal wash, or other lavage or washing). In some embodiments, the biological sample comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as the context will indicate, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of certain components, etc.

[0121] The term "cancer stage" refers to a qualitative or quantitative assessment of the level of progression of cancer. In some embodiments, criteria used to determine the stage of cancer may include, but are not limited to, one or more of the following: where the cancer is located in the body, tumor size, whether the cancer has spread to lymph nodes, whether the cancer has spread to one or more different parts of the body, etc. In some embodiments, cancer may be staged using the so-called TNM system, where T refers to the size and extent of the main tumor, usually called the primary tumor, N refers to the number of nearby lymph nodes that have cancer, and M refers to whether the cancer has metastasized. In some embodiments, cancer may be referred to as stage 0 (abnormal cells present without spreading to nearby tissues, also called carcinoma in situ or CIS; CIS is not cancer, but has the potential to become cancer), stages I-III (cancer is present; the higher the number, the larger the tumor and the more it has spread to nearby tissues), or stage IV (cancer has spread to distant parts of the body). In some embodiments, the cancer may be assigned a stage selected from the group consisting of: normal location; localized (cancer is confined to where it began and has no signs of spread); regional (cancer has spread to nearby lymph nodes, tissues, or organs); distant (cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the stage).

[0122] The phrase "therapeutic agent" may refer to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a model organism or a population of human subjects. In some embodiments, the appropriate population may be defined by various criteria, such as a certain 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, ameliorate, mitigate, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. 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 sold for administration to humans. In some embodiments, a therapeutic agent is an agent that requires a prescription for administration to humans.

[0123] Various aspects of the present disclosure are described in further detail in the following subsections. The present disclosure provides antigen-binding systems and binding agents comprising at least an anti-CD20 binding motif. Among other things, the present disclosure provides methods and compositions useful for treating cancer and / or initiating or modulating an immune response. In certain embodiments, the present disclosure includes antigen-binding systems and binding agents that are dual-targeted by comprising an anti-CD20 binding motif and a second binding motif for a second antigen or epitope. In some examples, the second binding motif selectively binds to CD19. In various embodiments, one or more binding motifs is an scFv. The amino acid sequences of exemplary binding motifs and nucleic acid sequences encoding the amino acid sequences are provided herein. In some embodiments, the antigen-binding systems of the present disclosure are chimeric antigen receptors. In some In embodiments, the antigen binding system of the present disclosure is a bispecific chimeric antigen receptor or a bicistronic chimeric antigen receptor. In some embodiments, the binding agent of the present disclosure is an engineered T cell receptor.

[0124] Various embodiments of the present disclosure provide vectors encoding the binding motifs or antigen-binding systems described 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 that are cells encoding or expressing the antigen-binding systems or binding motifs described 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, including, for example, binding agents that are immune cells genetically modified with an integrated gene, e.g., a nucleotide sequence of interest (e.g., a constitutive expression construct and / or an inducible expression construct comprising such a nucleotide sequence). In some embodiments, the present disclosure provides methods of treating a subject having a tumor, comprising administering to the subject a binding agent therapeutic described herein and / or a protein therapeutic described herein. In some embodiments, the method further comprises administering one or more additional therapeutic agents (e.g., a second binding agent (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 bispecific antibody that binds to T cells, an engineered antibody, and / or a polypeptide described herein).

[0125] Other features, objects, and advantages of the present disclosure will be apparent from the following detailed description, which should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, not limitation.

[0126] An anti-CD20 binding motif of the present disclosure may comprise an antigen-binding sequence found in an antibody described herein. In some cases, an anti-CD20 binding motif of the present disclosure comprises an antigen-binding fragment described herein. Unless otherwise indicated, reference to CD20 in this disclosure should be understood to relate to human CD20. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one heavy chain CDR (HCDR) shown herein, e.g., at least one HCDR disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two HCDRs shown herein, e.g., at least two HCDRs disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three HCDRs shown herein, e.g., three HCDRs disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one light chain CDR (LCDR) shown herein, e.g., at least one LCDR disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two LCDRs shown herein, e.g., at least two LCDRs disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three LCDRs shown herein, e.g., three LCDRs disclosed in any one of Tables 4-13.

[0127] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one HCDR shown herein, e.g., at least one HCDR disclosed in any one of Tables 4-13, and at least one LCDR shown herein, e.g., at least one LCDR disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one HCDR shown herein, e.g., at least one HCDR disclosed in any one of Tables 4-13, and at least one LCDR shown herein. For example, an anti-CD20 binding motif of the present disclosure comprises two HCDRs as shown herein, e.g., at least two HCDRs disclosed in any one of Tables 4-13, and two LCDRs as shown herein, e.g., at least two LCDRs disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two HCDRs as shown herein, e.g., at least two HCDRs disclosed in any one of Tables 4-13, and two LCDRs as shown herein, e.g., derived from the same table as the HCDR(s) in Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three HCDRs shown herein, e.g., three HCDRs disclosed in any one of Tables 4-13, and three LCDRs shown herein, e.g., three LCDRs disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three HCDRs shown herein, e.g., three HCDRs disclosed in any one of Tables 4-13, and three LCDRs derived from the same table as the HCDR(s) of Tables 4-13.

[0128] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises 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 of a heavy chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13.

[0129] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13.

[0130] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in any one of Tables 4-13, and at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in any one of Tables 4-13, and one light chain FR of a light chain variable domain disclosed herein, e.g., derived from the same table as the heavy chain FR(s) of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13, and two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in any one of Tables 4-13, and two light chain FRs of a light chain variable domain disclosed herein. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three heavy chain FRs from a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs from a heavy chain variable domain disclosed in any one of Tables 4-13, and three light chain FRs from a light chain variable domain disclosed herein, e.g., three light chain FRs from a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises three heavy chain FRs from a heavy chain variable domain disclosed herein, e.g., three light chain FRs from a light chain variable domain disclosed in any one of Tables 4-13, and three light chain FRs derived from the same table as the heavy chain FR(s) in Tables 4-13.

[0131] The exemplary antibody sequences shown in Tables 4-13 are suitable for use in any antibody format, including, for example, tetrameric antibodies, monospecific antibodies, bispecific antibodies, antigen-binding fragments, or binding motifs. The heavy and light chain variable domains and portions thereof shown in Tables 4-13 can be included in a binding motif.

[0132] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises one, two, or three FRs that, together or individually, have at least 75% (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) identity to the corresponding FR(s) of a heavy chain variable domain disclosed in any one of Tables 4 to 13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises one, two, or three FRs that, together or individually, have at least 75% (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%) identity to the corresponding FR(s) of a light chain variable domain disclosed in any one of Tables 4 to 13.

[0133] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a heavy chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to a heavy chain variable domain disclosed in Tables 4-13, and these heavy chain variable domains can be the same or different.

[0134] In various embodiments, an anti-CD20 binding motif of the present disclosure comprises at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a light chain variable domain disclosed in any one of Tables 4-13. In various embodiments, an anti-CD20 binding motif of the present disclosure comprises 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%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a light chain variable domain disclosed in any one of Tables 4 to 13, and these light chain variable domains can be the same or different.

[0135] In various embodiments, the anti-CD20 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) 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%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a light chain variable domain disclosed in any one of Tables 4 to 13. In certain embodiments, an anti-CD20 binding motif of the present disclosure comprises 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%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a heavy chain variable domain disclosed in any one of Tables 4 to 13, and 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%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a heavy chain variable domain disclosed in any one of Tables 4 to 13. and 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%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to one of the light chain variable domains disclosed in Tables 4 to 13, wherein the heavy chain variable domain and the light chain variable domain are optionally derived from the same table, Tables 4 to 13.

[0136] In various embodiments, the anti-CD20 binding motif of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a heavy chain variable domain disclosed in 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) to a light chain variable domain disclosed in Tables 4-13. and two light chain variable domains each having an identity of at least 0%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%), wherein in various embodiments (i) each of the heavy chain variable domains can be the same or different, (ii) each of the light chain variable domains 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, Tables 4 to 13, or (iv) two heavy chain variable domains and two light chain variable domains can all be derived from the same table, Tables 4 to 13. Each of Tables 4-13 shows the sequences of the heavy and light chain variable domains of an exemplary antibody, including (i) the heavy chain variable domain of an exemplary antibody, (ii) a DNA sequence encoding the heavy chain variable domain, (iii) three heavy chain variable domain CDRs of the heavy chain variable domain according to the IMGT, Kabat, and Chothia numbering systems, (iv) the light chain variable domain of an exemplary antibody, (v) a DNA sequence encoding the light chain variable domain, and (vi) three light chain variable domain CDRs of the light chain variable domain according to the IMGT, Kabat, and Chothia numbering systems. The information shown in each table provides the framework amino acid sequence, as well as the nucleotide sequence encoding each CDR amino acid sequence and the nucleotide sequence encoding the corresponding FR amino acid sequence.

[0137] In various embodiments, the binding motif has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100%, e.g., 85% to 90%, to a heavy chain variable domain of the disclosure (e.g., a heavy chain variable domain of any one of Tables 4 to 13). , 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity to a light chain variable domain of the present disclosure (e.g., having at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity to a light chain variable domain of any one of Tables 4 to 13), and a linker (e.g., a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307 to 313; e.g., a linker according to Whitlow et al. al. Protein Eng. 1993 Nov;6(8):989-95). In various embodiments, the binding motif may comprise a leader sequence and a heavy chain variable domain of the disclosure (e.g., a heavy chain variable domain of any one of Tables 4-13 that has at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity). The variable domain may comprise a heavy chain variable domain of the present disclosure (having at least 75% sequence identity, e.g., at least 80%, 85%, 90%, 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity, to a light chain variable domain of any one of Tables 4 to 13), and a linker. When the amino acid sequence or nucleotide sequence of a binding motif comprising a heavy chain variable domain of the present disclosure and a light chain variable domain of the present disclosure is provided, the linker connecting the two variable domains will be clear from the sequence in light of the present disclosure. When the amino acid sequence or nucleotide sequence of a binding motif comprising a heavy chain variable domain of the present disclosure and a light chain variable domain of the present disclosure is provided, the leader sequence will be clear in light of the present disclosure. For the avoidance of doubt, the heavy and light chain variable domains of the present disclosure may be present in any orientation, for example, with the heavy chain variable domain C-terminal to the light chain variable domain, or with the heavy chain variable domain N-terminal to the light chain variable domain. In various embodiments, the binding motif may comprise a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307 to 313, flanked by one or more additional linkers.

[0138] In certain embodiments, an anti-CD20 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to SEQ ID NO: 247. In certain embodiments, an anti-CD20 binding motif of the present disclosure comprises 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 to 313. In certain embodiments, an anti-CD20 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to SEQ ID NO: 245. In certain embodiments, an anti-CD20 binding motif of the present disclosure comprises a binding motif comprising a CSF2RA leader sequence according to SEQ ID NO: 245. In certain embodiments, an anti-CD20 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, a linker of the present disclosure, and a leader sequence of the present disclosure. Exemplary nucleotide sequences encoding anti-CD19 binding motifs and components thereof are found in SEQ ID NO: 246 and SEQ ID NO: 248. In various embodiments, a binding motif of the present disclosure has a sequence according to any one of the sequences in Table 53 (SEQ ID NO: 251-SEQ ID NO: 260).

[0139] Binding agents of the present disclosure based on the exemplary antibodies provided herein, such as Ab1, include: It may be provided in any fragment or format that includes a heavy chain variable domain according to the exemplary antibody shown and a light chain variable domain according to the exemplary antibody shown.

[0140] TIFF2025169340000004.tif254170TIFF2025169340000005.tif122170

[0141] TIFF2025169340000006.tif254170TIFF2025169340000007.tif128170

[0142] TIFF2025169340000008.tif254170TIFF2025169340000009.tif127170

[0143] TIFF2025169340000010.tif254170TIFF2025169340000011.tif126170

[0144] TIFF2025169340000012.tif254170TIFF2025169340000013.tif132170

[0145] TIFF2025169340000014.tif254170TIFF2025169340000015.tif132170

[0146] TIFF2025169340000016.tif254170TIFF2025169340000017.tif131170

[0147] TIFF2025169340000018.tif254170TIFF2025169340000019.tif147170

[0148] TIFF2025169340000020.tif254170TIFF2025169340000021.tif147170

[0149] TIFF2025169340000022.tif254170TIFF2025169340000023.tif142170

[0150] The present disclosure includes antibodies and antigen-binding systems comprising an anti-CD20 binding motif and a second binding motif that binds to a second target antigen or epitope, such as an antigen that is not CD20 (e.g., CD19). Dual-targeting antigen-binding systems include bispecific CARs and bicistronic CARs. Many antigen-binding motifs are known. In various embodiments, the second target antigen is CD19. The present disclosure includes, but is 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, HER1-H, and the like. and a variety of second target antigens, including a second antigen that is an ER2 combination, a HER2-HER3 combination, HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Rα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, or a combination thereof. Thus, in various embodiments, the antigen binding system or antibody of the disclosure comprises a first binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif and a second binding motif that is an anti-CD20 binding motif. and a second binding motif that binds to a second antigen that is ER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Rα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B-cell activating factor receptor), or a combination thereof. In some embodiments, the second antigen is an antigen characteristic of B cells or a subset thereof, and optionally, the second antigen is not CD19 or CD20. Examples of binding motifs that target these second antigens are known and / or provided herein.

[0151] 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 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 its heavy chain variable domain and / or light chain variable domain).

[0152] CD19 (also known as cluster of differentiation 19, B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, B4, CVID3, differentiation antigen CD19) is a protein encoded by the CD19 gene in humans. Unless otherwise indicated, references to CD19 in this disclosure should be understood to relate to human CD19. CD19 is found on the surface of B cells. Because expression of CD19 is a hallmark of B cells, CD19 may be useful as an antigen, for example, in recognizing B cells and cancer cells arising from B cells, such as B-cell lymphomas. Anti-CD19 antibodies can bind to CD19 expressed, for example, on B lymphocytes in peripheral blood and spleen, B-cell chronic lymphocytic leukemia (B-CLL) cells, prolymphocytic leukemia (PLL) cells, hairy cell leukemia (HCL) cells, common acute lymphoblastic leukemia (CALL) cells, pre-B-cell acute lymphoblastic leukemia (pre-B-ALL) cells, and null-cell acute lymphoblastic leukemia (NULL-ALL) cells, to name a few non-limiting examples. An exemplary pharmaceutical product comprising an antigen-binding system containing an anti-CD19 binding motif is the pharmaceutical product YESCARTA™. YESCARTA™ is a CD19-directed genetically modified autologous T-cell immunotherapeutic 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 the FDA-approved package insert for YESCARTA™, which is incorporated herein by reference in its entirety, for methods and compositions related to immunotherapy). Another exemplary pharmaceutical product comprising an antigen-binding system containing an anti-CD19 binding motif is the pharmaceutical product KYMRIAH™.KYMRIAH™ is a CD19-directed genetically modified autologous T-cell immunotherapy indicated for the treatment of (1) patients up to 25 years of age with precursor B-cell acute lymphoblastic leukemia (ALL) that is refractory or in second or subsequent relapse, 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 not otherwise specified (DLBCL), high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma (see the FDA-approved package insert for KYMRIAH™, which is incorporated herein by reference in its entirety, regarding methods and compositions related to immunotherapy).

[0153] Both YESCARTA™ and KYMRIAH™ contain antibody binding domains derived from anti-human CD19 antibodies. Many anti-CD19 antibodies are believed to bind to an epitope of CD19 encoded by exon 4 of the CD19 gene. Other anti-CD19 binding motifs may recognize different epitopes of CD19, or the same epitope with different affinities. The antigen binding system may, for example, contain an antigen binding domain derived from SJ25C1. The CD19 antibody clone SJ25C1 was obtained by 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 has been used in other investigational CD19-targeted chimeric antigen receptor (CAR) T-cell therapies.

[0154] The anti-CD19 binding motifs of the present disclosure may comprise antigen-binding sequences found in the antibodies described herein. In some embodiments, the anti-CD19 binding motifs of the present disclosure comprise antigen-binding fragments shown herein.

[0155] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one HCDR shown herein, e.g., at least one HCDR disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two HCDRs shown herein, e.g., at least two HCDRs disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises three HCDRs shown herein, e.g., the three HCDRs disclosed in Table 14.

[0156] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one LCDR shown herein, e.g., at least one LCDR disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two LCDRs shown herein, e.g., at least two LCDRs disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises three LCDRs shown herein, e.g., three LCDRs disclosed in Table 14.

[0157] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one HCDR shown herein, e.g., at least one HCDR disclosed in Table 14, and at least one LCDR shown herein, e.g., at least one LCDR disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two HCDRs shown herein, e.g., at least two HCDRs disclosed in Table 14, and two LCDRs shown herein, e.g., at least two LCDRs disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises three HCDRs shown herein, e.g., the three HCDRs disclosed in Table 14, and three LCDRs shown herein, e.g., the three LCDRs disclosed in Table 14.

[0158] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises 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 of a heavy chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in Table 14.

[0159] In various embodiments, the anti-CD19 binding motif of the present disclosure comprises a light chain In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in Table 14.

[0160] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one heavy chain FR of a heavy chain variable domain disclosed herein, e.g., at least one heavy chain FR of a heavy chain variable domain disclosed in Table 14, and at least one light chain FR of a light chain variable domain disclosed herein, e.g., at least one light chain FR of a light chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., at least two heavy chain FRs of a heavy chain variable domain disclosed in Table 14, and two light chain FRs of a light chain variable domain disclosed herein, e.g., at least two light chain FRs of a light chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises three heavy chain FRs of a heavy chain variable domain disclosed herein, e.g., three heavy chain FRs of a heavy chain variable domain disclosed in Table 14, and three light chain FRs of a light chain variable domain disclosed herein, e.g., three light chain FRs of a light chain variable domain disclosed in Table 14.

[0161] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises one, two, or three FRs that, together or each individually, have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to the corresponding FR(s) of a heavy chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises one, two, or three FRs that, together or each individually, have at least 75% identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to the corresponding FR(s) of a light chain variable domain disclosed in Table 14.

[0162] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a heavy chain variable domain disclosed in Table 14. In various embodiments, an anti-CD19 binding motif of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to a heavy chain variable domain disclosed in Table 14, and these heavy chain variable domains can be the same or different.

[0163] In various embodiments, the anti-CD19 binding motif of the present disclosure comprises at least one light chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to a light chain variable domain disclosed in Table 14. In embodiments, an anti-CD19 binding motif of the present disclosure comprises 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%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to a light chain variable domain disclosed in Table 14, and these light chain variable domains can be the same or different.

[0164] In various embodiments, an anti-CD19 binding motif of the present disclosure comprises at least one heavy chain variable domain having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) 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%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to a light chain variable domain disclosed in Table 14.

[0165] In various embodiments, the anti-CD19 binding motif of the present disclosure comprises two heavy chain variable domains each having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to a heavy chain variable domain disclosed in Table 14, and at least and two light chain variable domains each having 75% sequence 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% identity), where in various embodiments (i) each of the heavy chain variable domains can be the same or different, or (ii) each of the light chain variable domains can be the same or different.

[0166] In certain embodiments, an anti-CD19 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a linker having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identity) to SEQ ID NO: 247. In certain embodiments, an anti-CD19 binding motif of the present disclosure comprises 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 to 313. In certain embodiments, an anti-CD19 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, and a leader sequence having at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100%, e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100% identity) to SEQ ID NO: 245. In certain embodiments, an anti-CD19 binding motif of the present disclosure comprises a binding motif comprising a CSF2RA leader sequence according to SEQ ID NO: 245. In certain embodiments, an anti-CD19 binding motif of the present disclosure comprises a binding motif comprising a heavy chain variable domain of the present disclosure, a light chain variable domain of the present disclosure, a linker of the present disclosure, and a leader sequence of the present disclosure. In certain embodiments, the binding motif has the sequence set forth in SEQ ID NO: 243. Exemplary nucleotide sequences encoding the anti-CD19 binding motif and its components are found in SEQ ID NO: 244, SEQ ID NO: 246, and SEQ ID NO: 248. In various embodiments, the binding motif is It may comprise a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307 to 313, flanked by one or more additional linkers.

[0167] TIFF2025169340000024.tif254170TIFF2025169340000025.tif254170TIFF2025169340000026.tif174170

[0168] Examples of antigen-binding systems include bispecific chimeric antigen receptors (CARs) and bicistronic chimeric antigen receptors (CARs). The present disclosure provides, inter alia, antigen-binding systems that target both CD20 and a second target antigen, e.g., CD19. In some embodiments, the antigen-binding systems of the present disclosure include bispecific antigen-binding systems. In some embodiments, the antigen-binding systems of the present disclosure include bicistronic antigen-binding systems (e.g., systems comprising a first CAR and a second CAR, where the first CAR and the second CAR are expressed in the same cell). A bicistronic CAR may include two CARs that bind to different targets encoded by a single vector. A bicistronic CAR 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 combinations of features shown in this example are exemplary and not limiting. In some embodiments, the first CAR and the second CAR of a bicistronic CAR (e.g., an anti-CD20 CAR and an anti-CD19 CAR) In some embodiments, the first CAR and the second CAR (e.g., an anti-CD20 CAR and an anti-CD19 CAR) of a bicistronic CAR are encoded by separate genes and / or expressed as separate mRNA molecules. The first CAR and the second CAR of a bicistronic CAR are encoded by and / or expressed together in a single mRNA molecule, wherein the expressed protein comprises the first CAR, a cleavable linker domain, and a second CAR. The first CAR and the second CAR of a bicistronic CAR are generally expressed together in immune cells, e.g., CAR-T cells, such that each CAR-T cell expresses a CAR targeting each of the target antigens (e.g., CD20 and CD19, respectively).

[0169] 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 comprises a first CAR and a second CAR, wherein the sequences of the first CAR and the second CAR differ only in the binding motif. In various embodiments, the bicistronic CAR comprises a first CAR and a second CAR, wherein the sequences of the first CAR and the second CAR differ only in the heavy chain variable domain sequence and / or the light chain variable domain sequence. Thus, in some embodiments, the first CAR and the second CAR of a bicistronic CAR can have the same or different sequences for one or more components thereof, for example, any or all of the same or different costimulatory domains. For example, one or both of the first and second CARs of a bicistronic CAR can comprise a costimulatory domain as set forth herein, such as a CD28 costimulatory domain, a 41BB costimulatory domain, an OX40 costimulatory domain, or an ICOS costimulatory domain.

[0170] The CAR of a bicistronic CAR may comprise a binding motif, a hinge, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The binding motif may be the anti-CD19 binding motif or the anti-CD20 binding motif of the present disclosure. 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 costimulatory domain or a 41BB costimulatory domain. The activation domain may be a CD3z activation domain.

[0171] In some embodiments, the first and second binding motifs (e.g., separate anti-CD20 and anti-CD19 binding motifs) are both contained in a single bispecific CAR. In such bispecific CARs, the CAR molecule itself can be engineered to recognize more than one antigen. In tandem bispecific CARs, the first and second binding motifs are extracellular and can be characterized as membrane-proximal and membrane-distal binding motifs. 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.

[0172] Chimeric antigen receptors (CARs) are engineered receptors that can induce or redirect T cells (e.g., patient T cells or donor T cells) to target a selected antigen. CARs can be engineered to recognize an antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. CARs generally contain an extracellular binding motif (e.g., an anti-CD20 binding motif and / or an anti-CD19 binding motif) that mediates antigen binding, a transmembrane domain that spans the cell membrane or is understood to span the cell membrane if the antigen-binding system is present on the cell surface or membrane, and an intracellular (or cytoplasmic) signaling domain.

[0173] According to at least one non-limiting aspect, there are at least three "generations" of CAR compositions. In the first generation CARs, the binding motif (e.g., a single-chain fragment) is The variable binding motif (VBA) is linked or connected to a signaling domain (e.g., CD3ζ) via a transmembrane domain, optionally including a hinge domain and one or more spacers. In second-generation CARs, a costimulatory domain (CM1, e.g., CD28, 4-1BB, or OX-40) is introduced along with the signaling domain (e.g., CD3ζ). In third-generation CARs, a second costimulatory domain (CM2) is included.

[0174] TCRs are heterodimers composed of an α chain and a β chain. TCR signaling requires the recruitment of signaling proteins to generate an immune synapse. Furthermore, the localization of the TCR at the plasma membrane depends on the CD3 complex expressed in the T cell. Engineered single-chain TCRs can be generated, for example, using the transmembrane and signaling domains of a CAR construct. Methods and constructs for this are known (e.g., sTCR and TCR-CAR molecules, e.g., fusions of the TCR β chain with CD28TM and CD28 and CD3ζ signaling modules). The anti-CD20 and / or anti-CD19 antigen binding systems of the present disclosure can comprise one or more antigen-binding motifs that bind to CD20 and / or CD19. In some embodiments, the antigen binding system further comprises a costimulatory domain, and / or an extracellular domain (e.g., a "hinge" or "spacer" region), and / or a transmembrane domain, and / or an intracellular (signaling) domain, and / or a CD3ζ or CD3ε activation domain. In some embodiments, the anti-CD20 antigen-binding system and / or the 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 CD3ζ or CD3ε activation domain.

[0175] In some embodiments, an antigen binding system of the present disclosure may include an antigen binding system comprising one or more or all of a leader peptide (P), a binding motif (B), an extracellular domain of a costimulatory protein (E), a transmembrane domain (T), a costimulatory domain (C), a second costimulatory domain (C'), and an activation domain (A). In some cases, the antigen binding system is configured according to the following: BETA. In some cases, the antigen binding system is configured according to the following: PBETA. In some cases, the antigen binding system is configured according to the following: BETCA. In some cases, the antigen binding system is configured according to the following: PETCA. In some cases, the antigen binding system is configured according to the following: BETC C'A. In some embodiments, the antigen binding system comprises a VH and a VL, and optionally the CAR is configured according to the following: P-VH-VL-ETCA, or P-VL-VH-ETCA. In some embodiments, the VH and VL are connected by a linker (L), and optionally 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.

[0176] One or more antigen-binding motifs determine the target(s) of the antigen-binding system. The binding motif of the antigen-binding system can include any binding motif, e.g., an antibody provided by the present disclosure, e.g., a binding motif of the present disclosure. In some embodiments, the binding motif 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.

[0177] Binding motifs are used at least in part in chimeric antigen receptors, as they can be engineered to be expressed as part of a single chain together with other CAR components. See, for example, U.S. Patent Nos. 7,741,465 and 6,319,494, as well as 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 (626-630); 98, 161: 2791-2797 (each of which is incorporated herein by reference with respect to binding motif domains in CARs). A binding motif or scFv is a single-chain antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain and the light chain variable domain are linked or connected together. See, e.g., U.S. Pat. Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136 (each of which is incorporated herein by reference with respect to binding motif domains in CARs). When derived from a parent antibody, the binding motif may retain some, all, or essentially the binding of the parent antibody for the target antigen.

[0178] The hinge may be an extracellular domain of an antigen-binding system located between the binding motif and the transmembrane domain. The hinge may also be referred to as an extracellular domain or a "spacer." The hinge may contribute to the expression, activity, and / or stability of the receptor. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain. The hinge may also provide flexibility to reach the target antigen. The hinge comprises an immunoglobulin-like hinge domain.

[0179] In some embodiments, the antigen-binding system of the present disclosure may include a hinge that is, is derived from, or is derived from (e.g., including all or a fragment thereof) an immunoglobulin-like hinge domain. In some embodiments, the hinge domain is derived from or from an immunoglobulin. In some embodiments, the hinge domain is selected from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM hinge, or a fragment thereof.

[0180] The hinge may be derived from natural or synthetic sources. In some embodiments, the antigen binding system of the present disclosure comprises a binding site for one or more of the following antigens: CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA5), CD49g (ITGA6), CD49h (ITGA7), CD49i (ITGA8), CD49m (ITGA9), CD49m (ITGA10), CD49m (ITGA11), CD49m (ITGA12), CD49m (ITGA13), CD49m (ITGA14), CD49m (ITGA15), CD49m (ITGA16), CD49m (ITGA17), CD49m (ITGA18), CD49m (ITGA19 ... TGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2D L1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3ζ), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD 272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337(NK-p30), CD352(SLAMF6), CD35 3 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ,IL-7Rα, LFA-1, SLAMF9, LAT, GADS(GrpL), SLP-76(L, CP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or may comprise a hinge that is derived from or is derived from (e.g., including all or a fragment thereof), or is a fragment or combination thereof. In certain embodiments, the CAR does not comprise a CD28 hinge.

[0181] In some embodiments, the antigen binding system of the present disclosure may include a hinge that is, is derived from, or is derived from (e.g., including all or a fragment thereof) the hinge of CD8α. In some embodiments, the hinge is, is derived from, or is derived from the hinge of CD28. In some embodiments, the hinge is, is derived from, or is derived from a fragment of the hinge of CD8α or a fragment of the hinge of CD28, where a fragment is less than the whole. In some embodiments, the fragment of a CD8α hinge or a fragment of a CD28 hinge comprises an amino acid sequence excluding at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, or at least 20 amino acids from the N-terminus or C-terminus, or both, of a CD8α hinge or CD28 hinge. Exemplary hinge sequences include the hinge sequences set forth in Table 54 (SEQ ID NOs:261 to 269).

[0182] The polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, a polynucleotide encoding a hinge domain comprises a nucleotide 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.

[0183] Generally, a "transmembrane domain" (e.g., of an antigen binding system) refers to a domain that has the property of being intramembrane when present in a molecule on the surface of a cell or in the cell membrane (e.g., spanning part or all of the cell membrane). The costimulatory domain for the antigen binding system of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. It is not necessary for all amino acids in a transmembrane domain to be present within the membrane. For example, in some embodiments, a transmembrane domain is characterized by a specified stretch or portion of a protein being located substantially within the membrane. Amino acid or nucleic acid sequences can be analyzed using various algorithms that predict the subcellular localization (e.g., transmembrane localization) of proteins. The programs psort (PSORT.org) and Prosite (prosite.expasy.org) provide such programs. This is an example of a program.

[0184] The types of transmembrane domains included in the antigen binding systems described herein may be of any type. In some embodiments, a transmembrane domain is selected that is naturally associated with a binding motif and / or an intracellular domain. In some cases, the transmembrane domain contains one or more amino acid modifications (e.g., deletions, insertions, and / or substitutions) to minimize interactions with other members of the receptor complex, e.g., to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins.

[0185] Transmembrane domains can be derived from either natural or synthetic sources. If natural in origin, the domain can be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains include the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD45, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activation NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD1 9a, 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), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell costimulatory factor (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, L IGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a / 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 lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A;The transmembrane domain may be derived from (e.g., may comprise at least the transmembrane domain of) Ly108, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof. In some embodiments, the transmembrane domain may be synthetic (and may comprise primarily hydrophobic residues, e.g., leucine and valine). In some embodiments, a synthetic transmembrane domain includes a phenylalanine, tryptophan, and valine triplet at each end. In some embodiments, the transmembrane domain is directly linked or connected to the cytoplasmic domain. In some embodiments, a short oligopeptide or polypeptide linker (e.g., between 2 and 10 amino acids in length) may form the link between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.

[0186] The polynucleotide and polypeptide sequences of the transmembrane domains set forth herein are known. In some embodiments, the polynucleotide encoding the transmembrane domain comprises a nucleotide 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 the known nucleotide sequence. In some embodiments, the polypeptide sequences of the transmembrane domains are The sequence comprises a polypeptide sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, or 95%-100%) identical to a known polypeptide sequence. Optionally, a short spacer can form a link between all or a portion of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0187] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains that initiate and / or mediate an intracellular signal that, upon binding of a target antigen to the binding motif, activates, for example, one or more immune cell effector functions (e.g., native immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates activation of at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including cytokine secretion. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and / or other T cell function. The intracellular domain can comprise a signaling domain that is an activation domain. The intracellular domain can comprise a signaling domain that is a costimulatory signaling domain.

[0188] Intracellular signaling domains that can transmit signals when an antigen binds to an immune cell are known, and any of these can be included in the antigen-binding system of the present disclosure. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction after the TCR binds to an antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0189] In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs that contain cytoplasmic signaling sequences include those derived from TCRζ, FcRγ, FcRβ, CD3ζ, CD3γ, CD3δ, CD3ε, 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)).

[0190] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, I TGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD11a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, S Examples include ELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof.

[0191] For example, including a costimulatory signaling domain in a CAR can enhance signal transduction capabilities. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (see above), 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). Signals generated by the TCR alone may be insufficient to fully activate T cells, and secondary or costimulatory signals can enhance activation. Thus, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., costimulatory signaling domains) that activate one or more immune cell effector functions (e.g., native immune cell effector functions described herein). In some embodiments, a portion of such a costimulatory signaling domain may be used so long as that portion transmits an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell coreceptor (or fragment thereof). Non-limiting examples of such T cell coreceptors include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds CD83. An exemplary costimulatory protein has the amino acid sequence of a costimulatory protein found naturally on T cells, the complete native amino acid sequence of which is set forth in NCBI Reference Sequence: NP_006130.1. In certain instances, the CAR comprises a 41BB costimulatory domain encoded by a sequence according to SEQ ID NO: 270, as shown below.

[0192] SEQ ID NO: 270 AGATTCAGCGTTGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAG GAGGGCGGCTGTGAGCTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGAGGAATACGATGTGCTGG ACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGA AGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGATTAATTAAatcgat

[0193] The polynucleotide and polypeptide sequences of the signaling domains set forth herein are known. In some embodiments, a polynucleotide encoding a signaling domain comprises a nucleotide 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 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 99% identical to a known polypeptide sequence. The polypeptide sequences of the present invention may be at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) identical.

[0194] In various embodiments, mechanisms to modulate (e.g., decrease) the activity of an antigen binding system are desirable, for example, to minimize or reduce adverse events resulting from the activity of the antigen binding system. The inclusion of an inducible "on" switch or "accelerator" switch in immune cells can also be beneficial. It may also be desirable to transduce the cells with the CAR construct of the present disclosure. Suitable techniques include using inducible caspase-9 (U.S. Patent Application Publication No. 2011 / 0286980) or thymidine kinase before, after, or simultaneously with transduction of the cells with the CAR construct of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques.

[0195] According to the present disclosure, on-off control switch technologies or other types of control switch technologies can be part of the present specification. These technologies include, for example, those described in Wu et al., Science 2014 350 (6258) (their application), which utilize the FKBP / Rapalog dimerization system in certain cells. (The contents of which are incorporated herein by reference in their entireties.) Additional dimerization techniques are described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336 and U.S. Pat. Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the contents of each of which are also incorporated herein by reference with respect to dimerization techniques. Additional dimerization pairs may include cyclosporin-A / cyclophilin receptor, estrogen / estrogen receptor (optionally using tamoxifen, 4-hydroxytamoxifen, or endoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, and / or vitamin D / vitamin D receptor. Further examples of dimerization techniques can be found, for example, in WO 2014 / 127261, WO 2015 / 090229, U.S. Patent Application Publication No. 2014 / 0286987, U.S. Patent Application Publication No. 2015 / 0266973, U.S. Patent Application Publication No. 2016 / 0046700, U.S. Patent No. 8,486,693, U.S. Patent Application Publication No. 2014 / 0171649, and U.S. Patent Application Publication No. 2012 / 0130076, the contents of which are further incorporated by reference in their entireties.

[0196] In some embodiments, the antigen binding system of the present disclosure comprises 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).

[0197] Components of a CAR can be exchanged or "swapped" for equivalent components using routine techniques in biotechnology. To provide a few non-limiting and partial examples, a CAR of the present disclosure can include a binding motif shown herein in combination with a hinge shown herein and a costimulatory domain shown herein. In certain examples, a CAR of the present disclosure can include a binding motif shown herein with a leader sequence shown herein in combination with a hinge shown herein and a costimulatory domain shown herein. In various embodiments, the present disclosure provides a binding motif according to any one of SEQ ID NOs: 251-260 in combination with (e.g., adjacently fused to) a hinge according to any one of SEQ ID NOs: 261-269, optionally further combined with (e.g., adjacently fused to) a 41BB costimulatory domain according to SEQ ID NO: 270. A few non-limiting examples of these are shown in SEQ ID NOs: 271-290.

[0198] A bicistronic CAR can comprise a first CAR sequence and a second CAR sequence expressed as a single polypeptide with a cleavable linker between the first and second CARs. An exemplary cleavable linker is Furin-GSG-T2A (see, e.g., Chng et al. MAbs. 2015 Mar-Apr; 7(2): 403-412, which is incorporated by reference for information regarding cleavable linkers, and see, e.g., Chng et al. MAbs. 2015 Mar-Apr; 7(2): 403-412, which is incorporated by reference for information regarding bicistronic CAR design). (See Guedan et al. Mol Ther Methods Clin Dev. 2019 Mar 15; 12: 145-156, which is incorporated herein by reference.) To provide one non-limiting example, a bicistronic CAR may comprise: (a) a first CAR comprising (i) a signal peptide (e.g., CSF2RA signal peptide), (ii) an anti-CD19 light chain variable domain, (iii) a linker (e.g., a G4S linker or multiple G4S linkers), (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); and (b) a cleavable linker (e.g., a furin GSG-T2A linker), and (c) a second CAR comprising (i) a signal peptide (e.g., CD8a signal peptide), (ii) an anti-CD20 heavy chain variable domain of the present disclosure, (iii) a linker (e.g., a linker according to SEQ ID NO: 247 and / or a linker according to any one of SEQ ID NOs: 307-313), (iv) an anti-CD20 light chain variable domain of the present disclosure, (v) a spacer or hinge (e.g., a CD8a spacer), (vi) a transmembrane domain (e.g., a CD8 transmembrane domain), (vii) a costimulatory domain (e.g., a 41bb costimulatory domain), and (viii) a stimulatory domain (e.g., a CD3z stimulatory domain). Thus, without limitation, an exemplary anti-CD20 / anti-CD19 bicistronic CAR can have or include the nucleotide and amino acid sequences set forth in SEQ ID NOs: 291 and 292.

[0199] A bispecific CAR can be a single polypeptide comprising a first binding motif of the present disclosure and a second binding motif that is an anti-CD19 binding motif. In non-limiting exemplary embodiments, the bispecific CAR can comprise: (i) a leader (e.g., a CSF2RA signal peptide), (ii) an anti-CD20 light chain variable domain of the present disclosure, (iii) a linker, (iv) an anti-CD20 heavy chain variable domain, (v) a linker (e.g., a shortened linker), (vi) an anti-CD19 light chain variable domain, (vii) a linker, (viii) an anti-CD19 heavy chain variable domain, (ix) an extracellular domain (e.g., a CD28T hinge or an IgG4 hinge), (x) a transmembrane domain (e.g., a CD28 transmembrane domain), (xi) an intracellular region (e.g., a CD28 intracellular costimulatory domain and / or a 41bb costimulatory domain), and a stimulatory domain (e.g., a CD3z stimulatory domain). Thus, without limitation, an exemplary anti-CD20 / anti-CD19 bispecific CAR can have or include the nucleotide and amino acid sequences set forth in SEQ ID NO:293 to SEQ ID NO:306.

[0200] Various CAR sequences, components, and / or frameworks, including but not limited to sequences of hinges, spacers, transmembrane domains, costimulatory domains, stimulatory domains, binding motifs, and variants of each, are known, and CARs with desired binding and components or structures can be readily constructed, for example, given the heavy chain variable domain or CDR sequences and light chain variable domain or CDR sequences.

[0201] The present disclosure provides, among other things, bispecific antibodies that bind to CD20 and a second target antigen, e.g., CD19. Bispecific antibodies include antibodies 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 includes 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 includes 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.

[0202] The present disclosure includes conjugates in which an antibody of the present disclosure is linked to a therapeutic agent or detectable moiety. In various embodiments, the therapeutic agent is an anti-cancer agent as described herein. In certain embodiments, the conjugates described 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 are useful in diagnostic or imaging applications, for example, in diagnosing or imaging cancer. Any of a wide variety of detectable moieties can be used in the labeled antibody conjugates described herein. Suitable detectable moieties include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), microparticles, metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0203] The present disclosure includes nucleic acids encoding the anti-CD20 binding motif and / or anti-CD19 binding motif provided herein. The present disclosure includes nucleic acids encoding the antibodies provided herein, including, but not limited to, nucleic acids encoding binding motifs (e.g., anti-CD20 binding motif and anti-CD19 binding motif). The present disclosure includes nucleic acids encoding the antigen binding systems provided herein, including, but not limited to, nucleic acids encoding bicistronic chimeric antigen receptors and bispecific chimeric antigen receptors (e.g., bicistronic chimeric antigen receptors and bispecific chimeric antigen receptors that bind CD20 and CD19). The nucleic acid sequence of SEQ ID NO:2 corresponds to, and provides exemplary nucleic acid sequences encoding, SEQ ID NO:1 and SEQ ID NO:3-11, respectively. The nucleic acid sequence of SEQ ID NO:13 corresponds to, and provides exemplary nucleic acid sequences encoding, SEQ ID NO:12 and SEQ ID NO:14-22, respectively. The nucleic acid sequence of SEQ ID NO:24 corresponds to, and provides exemplary nucleic acid sequences encoding, SEQ ID NO:23 and SEQ ID NO:25-33, respectively. The nucleic acid sequence of SEQ ID NO:35 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:34 and SEQ ID NO:36-44, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:46 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:45 and SEQ ID NO:47-55, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:57 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:56 and SEQ ID NO:58-66, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:68 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:67 and SEQ ID NO:69-77, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:79 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:78 and SEQ ID NO:80-88, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:90 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:89 and SEQ ID NO:91-99, respectively, and is provided.The nucleic acid sequence of SEQ ID NO:101 includes and provides exemplary nucleic acid sequences that correspond to and encode SEQ ID NO:100 and SEQ ID NO:102 through SEQ ID NO:110, respectively. The nucleic acid sequence of SEQ ID NO:112 corresponds to SEQ ID NO:111 and SEQ ID NO:112. The nucleic acid sequence of SEQ ID NO: 123 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO: 122 and SEQ ID NO: 124-132, respectively. The nucleic acid sequence of SEQ ID NO: 134 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO: 133 and SEQ ID NO: 135-143, respectively. The nucleic acid sequence of SEQ ID NO: 145 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO: 144 and SEQ ID NO: 146-154, respectively. The nucleic acid sequence of SEQ ID NO: 156 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO: 155 and SEQ ID NO: 157-165, respectively. The nucleic acid sequence of SEQ ID NO: 167 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO: 166 and SEQ ID NO: 168-176, respectively. The nucleic acid sequence of SEQ ID NO:178 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:177 and SEQ ID NO:179-187, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:189 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:188 and SEQ ID NO:190-198, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:200 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:199 and SEQ ID NO:201-209, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:211 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:210 and SEQ ID NO:212-220, respectively, and is provided. The present disclosure includes nucleic acids encoding the anti-CD19 binding motifs set forth herein. The nucleic acid sequence of SEQ ID NO:222 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:221 and SEQ ID NO:223-231, respectively, and is provided. The nucleic acid sequence of SEQ ID NO:233 corresponds to and includes exemplary nucleic acid sequences encoding SEQ ID NO:232 and SEQ ID NO:234 ​​through SEQ ID NO:242, respectively, and is provided.

[0204] The present disclosure includes vectors comprising nucleic acids of the present disclosure and / or encoding polypeptides of the present disclosure. In various embodiments, the present disclosure includes vectors comprising nucleic acids encoding the anti-CD20 binding motif and / or anti-CD19 binding motif described herein. In various embodiments, the present disclosure includes vectors comprising nucleic acids encoding the antibodies described herein, including, but not limited to, nucleic acids encoding binding motif molecules (e.g., anti-CD20 binding motif or anti-CD19 binding motif). In various embodiments, the present disclosure includes vectors comprising nucleic acids encoding one or more antigen binding systems described herein, including, but not limited to, nucleic acids encoding bicistronic chimeric antigen receptors or bispecific chimeric antigen receptors (e.g., bicistronic chimeric antigen receptors and bispecific chimeric antigen receptors that bind CD20 and CD19).

[0205] Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, 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 T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0206] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and / or expression, such as plasmids and viruses. For example, vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), ), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series The expression vector may be selected from the following: λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149. Bacteriophage 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 (e.g., from Clontech) is used to contain both the nucleic acid encoding the antigen binding system and the inducible expression construct described herein.

[0207] In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alphavirus vectors, vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, and fowlpox virus vectors, and preferably have the natural or engineered ability to transform immune cells (e.g., T cells).

[0208] Recombinant expression vectors are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 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 contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0209] Recombinant expression vectors may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation that confers prototrophy in auxotrophic hosts, etc. Suitable marker genes for recombinant expression vectors include, for example, the neomycin / G418 resistance gene, the puromycin resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0210] Vectors useful in the context of the present disclosure can be "naked" nucleic acid vectors (i.e., vectors with little or no proteins, sugars, 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.

[0211] Vector DNA can be introduced into cells, e.g., immune cells, via conventional transformation, transfection, or transduction techniques. The terms "transformation" and "transfection" refer to methods such as calcium phosphate co-precipitation or calcium chloride co-precipitation, DEAE co-precipitation, and the like. - encompasses various art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into cells, such as dextran-mediated transfection, lipofection, gene guns, nanoparticle-mediated delivery, or electroporation. Transduction includes viral delivery of vectors into cells, for example, by vectors disclosed herein, including, but not limited to, retroviruses, lentiviruses, and AAV.

[0212] 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 the 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 described herein. The present disclosure includes, but is not limited to, cells that contain or are transformed to contain at least one vector encoding an antibody described herein, including a binding motif molecule (e.g., an anti-CD20 binding motif or an anti-CD19 binding motif). The present disclosure includes cells containing or transformed to contain at least one vector encoding one or more antigen binding systems described herein, including, but not limited to, a bicistronic chimeric antigen receptor or a bispecific chimeric antigen receptor (e.g., a bicistronic chimeric antigen receptor or a bispecific chimeric antigen receptor that binds CD20 and CD19). In some embodiments, cells are co-transfected or co-transduced with two vectors, each encoding a different CAR, and the two different CARs together form a bicistronic CAR. Transfection or transduction of cells with two different vectors encoding two different CARs that together form a bicistronic CAR can be performed simultaneously on a single cell population, simultaneously on two different cell populations, each transduced with only one of the two vectors, or independently on two different cell populations, each transduced with only one of the two vectors.

[0213] The present disclosure includes cells comprising one or more polypeptides of the present disclosure. The present disclosure includes cells comprising (e.g., expressing) the anti-CD20 binding motif and / or anti-CD19 binding motif described herein. The present disclosure includes cells comprising (e.g., expressing) the antibodies described herein, including, but not limited to, a binding motif (e.g., an anti-CD20 binding motif or an anti-CD19 binding motif). The present disclosure includes cells comprising (e.g., expressing) one or more antigen binding systems described herein, including, but not limited to, a bicistronic chimeric antigen receptor or a bispecific chimeric antigen receptor (e.g., a bicistronic chimeric antigen receptor and a bispecific chimeric antigen receptor that binds CD20 and CD19).

[0214] In other aspects, provided herein are cells comprising the polynucleotides or vectors of the present disclosure. In some embodiments, the present disclosure is directed to cells, e.g., in vitro cells, comprising a polynucleotide encoding a CAR or TCR comprising one or two scfvs disclosed herein. In other embodiments, the present disclosure is directed to cells, e.g., in vitro cells, comprising a polypeptide encoded by a CAR or TCR comprising one or two scfvs disclosed herein. In some embodiments, the polypeptide comprises the amino acid sequence shown below, or any combination thereof.

[0215] SEQ ID NO: 232 (anti-CD19 scFv light chain):

[0216] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0217] SEQ ID NO: 221 (anti-CD19 scFv heavy chain):

[0218] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0219] SEQ ID NO: 56 (anti-CD20 light chain):

[0220] DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

[0221] SEQ ID NO: 45 (anti-CD20 heavy chain):

[0222] QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETDYSSGMGYGMDVWGQGTTVTVSS

[0223] SEQ ID NO: 56 (anti-CD20 scFv2 light chain):

[0224] DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLADPFTFGGGTKVEIK

[0225] SEQ ID NO: 155 (anti-CD20 scFv2 heavy chain):

[0226] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSRYVWSWIRQPPGKGLEWIGEIDSSGKTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

[0227] SEQ ID NO: 144 (anti-CD20 scFv3 light chain):

[0228] DIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSFPWTFGGGTKVEIK

[0229] SEQ ID NO: 177 (anti-CD20 scFv3 heavy chain):

[0230] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYAWSWIRQPPGKGLEWIGEIDHRGFTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVRYDSSDSYYYSYDYGMDVWGQGTTVTVSS

[0231] SEQ ID NO: 78 (anti-CD20 scFv4 light chain):

[0232] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRFPPTFGQGTKVEIK

[0233] SEQ ID NO: 67 (anti-CD20 scFv4 heavy chain):

[0234] QVQLVQSGAEVKKPGASVKVSCKASGYTFKEYGISWVRQAPGQGLEWMGWISAYSGHTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGPHYDDWSGFIIWFDPWGQGTLVTVSS

[0235] Any cell can be used as a host cell for the polynucleotides, vectors, or polypeptides of the present disclosure. In some embodiments, the cell is a prokaryotic cell, a fungal cell, a yeast cell, or a yeast cell. or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, e.g., Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Klebsiella; Proteus; Salmonella, e.g., Salmonella Salmonella typhimurium; Serratia, e.g., Serratia marcescans and Shigella; B. subtilis Bacilli such as B. subtilis and B. licheniformis; P. aeruginosa and Pseudomonas, such as 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 (TILs), TCR-expressing cells, natural killer (NK) cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In certain embodiments, the T cell is a tumor-infiltrating lymphocyte (TIL), an autologous T cell, an engineered autologous T cell (eACT™), an allogeneic T cell, a xenogeneic T cell, or any combination thereof.

[0236] Chimeric antigen receptors (CARs or CAR-Ts) and engineered T cell receptors (TCRs) can be readily inserted into immune cells, e.g., T cells, and expressed by the immune cells, e.g., T cells, to produce binding agents. In certain embodiments, the cells (e.g., immune cells such as T cells) are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In other embodiments, the donor subject is a human patient not suffering from cancer or a tumor. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject.

[0237] The cells of the present disclosure can be obtained from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can 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. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using numerous techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In certain embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in a buffer or medium appropriate for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, washing steps can be performed using semi-automated flow-through centrifuges, such as the Cobe™ 2991 cell processing device, Baxter CytoMate™, and the like. 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, undesirable components of the apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0238] In certain embodiments, T cells are isolated from PBMS by lysis of red blood cells and depletion of monocytes, for example, by using centrifugation through a PERCOLL™ gradient. + , CD8 + , CD28 + , CD45RA + and CD45RO + Specific subpopulations of T cells, such as T cells from the IL-16 / ... This can be accomplished by a combination of antibodies against surface markers unique to the cells being negatively selected. In some embodiments, cell sorting and / or selection by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected can be used. For example, negative selection can result in CD4 + To enrich for 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.

[0239] In some embodiments, PBMCs are used directly for genetic modification with immune cells (such as CARs or TCRs) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes are further isolated and sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion, for both cytotoxic and helper T lymphocytes.

[0240] In some embodiments, CD8 + These various CD8 cells +By identifying cell surface antigens associated with the cells, they are further sorted into naive cells, central memory cells, and effector cells. In some embodiments, central memory T cells express phenotypic markers including CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and are negative for granzyme B. In some embodiments, central memory T cells express CD8 + , CD45RO + and CD62L + In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In certain embodiments, CD4 + T cells are further sorted into subpopulations, e.g., CD4 + Helper T cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations that have cell surface antigens.

[0241] In some embodiments, immune cells, e.g., T cells, are isolated and then genetically modified using known methods, or the immune cells are activated and expanded in vitro (i.e., in the case of progenitor cells, differentiated) prior to genetic modification. In another embodiment, immune cells, e.g., T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514, and PCT Publication WO 2012 / 079000, the contents of which are incorporated herein by reference in their entireties. Typically, such methods involve contacting PBMCs or isolated T cells with stimulators and costimulators, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same beads serve as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads™ system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and PCT Publication WO 2012 / 129514, the contents of which are incorporated herein by reference in their entireties.

[0242] In various aspects of the disclosure, vectors or polypeptides of the disclosure (e.g., anti-CD20 binding motifs and / or anti-CD20 / anti-CD19 antigen binding systems of the disclosure) are used to express or express the vectors or polypeptides of the disclosure (e.g., anti-CD20 binding motifs and / or anti-CD20 / anti-CD19 antigen binding systems of the disclosure). A cell that expresses, encodes, or is transformed to encode the same is a binding agent. A binding agent, or a population of binding agents, can be used as (e.g., as the active agent of) a binding agent (e.g., a composition comprising cells useful as a therapeutic agent for cancer).

[0243] The present disclosure further includes methods and processes for producing the antibody agents disclosed herein, for example, by transforming (e.g., transducing) a cell with a vector or nucleic acid of the present disclosure. In some embodiments, the method or process for producing an antibody agent disclosed herein comprises transforming (e.g., transducing) a cell with a nucleic acid (e.g., a nucleic acid present in a vector) encoding at least one antigen binding system described herein. Generally, the antibody agents described herein can be produced from immune cells, e.g., cells that are useful or can be used in adoptive cell therapy. In some embodiments, the binding agent is produced from TIL, T cells, CD8 + cells, CD4 + They are produced from a cell type selected from the group consisting of CD4 T cells, NK cells, γδ T cells, regulatory T cells, or peripheral blood mononuclear cells. "Tumor-infiltrating lymphocytes," or TILs, refer to white blood cells that have left the bloodstream and migrated to tumors. Lymphocytes can be divided into three groups: B cells, T cells, and natural killer cells. "T cells" are CD4 + Helper cells, CD8 + CD3, including cytotoxic T cells and γδ T cells + Refers to cells.

[0244] In certain embodiments, the binding agents are produced by genetically modifying (e.g., transforming) cells, e.g., immune cells, with nucleic acids and / or expression constructs encoding the antigen-binding systems described herein (e.g., (i) a first recombinant expression vector comprising a nucleic acid encoding the 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 the 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 nucleotide, including, but not limited to, DNA and RNA, which can be single-stranded or double-stranded, synthetic, or partially derived from natural sources, and can contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain linkages between naturally occurring or non-naturally occurring nucleotides, or both types of linkages.

[0245] In some embodiments, a method comprises transducing a cell with a polynucleotide encoding an antigen binding system disclosed herein, hi some embodiments, a method comprises transducing a cell with a vector comprising a polynucleotide encoding the antigen binding system.

[0246] In some embodiments, donor T cells used in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells used in T cell therapy are obtained from a subject who is not a patient. In an exemplary aspect, a subject can undergo leukapheresis, in which leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate cells of interest, e.g., T cells. These T cell isolates can be expanded by methods and treated such that one or more CAR constructs of the present disclosure can be introduced, thereby generating CAR T cells of the present disclosure.

[0247] In some embodiments, immune cells are obtained from a subject and transformed, e.g., transduced, with an expression vector comprising an inducible or constitutive expression construct described herein, e.g., an inducible or constitutive expression construct described herein, to obtain the binding agent. Thus, in some embodiments, the binding agent comprises autologous cells that are 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 expression vector comprising an inducible or constitutive expression construct described herein, e.g., an inducible or constitutive expression construct described herein, to obtain the binding agent. Transformation, eg, transduction, with an expression vector containing a constitutive or a synthetic expression construct results in a binding agent that can be allogeneically transplanted into another subject.

[0248] In certain embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient afflicted with cancer or a tumor. In other embodiments, the donor subject is a human patient not afflicted with cancer or a tumor.

[0249] Various compositions of the present disclosure include populations of engineered cells, which may be produced by any means. In some embodiments, the present disclosure provides populations of human cells engineered to express the antigen binding systems described herein. In some embodiments, such populations include binding agents. In some embodiments, such populations include cultured populations. In some embodiments, such populations are cultured populations of cells from a single human source, which may, in some embodiments, receive administration of the cultured population. As disclosed herein, binding agents can include any single cell or population of cells, for example, the populations of engineered cells described herein.

[0250] Other aspects of the present disclosure relate to compositions comprising the polynucleotides described herein, vectors described herein, polypeptides described herein, or in vitro cells described herein. In some embodiments, the compositions comprise a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some embodiments, the compositions comprise an excipient. In one embodiment, the composition comprises a polynucleotide encoding a CAR or TCR comprising an antigen-binding molecule described herein. In another embodiment, the composition comprises a CAR or TCR comprising a TCD encoded by a polynucleotide of the present disclosure. In another embodiment, the composition comprises a T cell comprising a CAR or TCR comprising one or two of the SCFVs disclosed herein.

[0251] In other embodiments, the composition is selected for parenteral delivery, for inhalation, or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of one of ordinary skill in the art. In certain embodiments, a buffer is used to maintain the composition at physiological pH or a slightly lower pH, typically in the range of about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, in which the composition described herein, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the preparation involves formulating the desired molecule with polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes that provide controlled or sustained release of the product, followed by delivery via depot injection. In certain embodiments, implantable drug delivery devices are used to introduce the desired molecule.

[0252] In some embodiments, the present disclosure provides pharmaceutical compositions comprising and / or delivering one or more of the present disclosure, e.g., an antigen binding system of the present disclosure, a nucleic acid encoding the antigen binding system, and / or a cell(s) or population of cells containing and / or expressing the nucleic acid.

[0253] In some embodiments, the present disclosure provides pharmaceutical compositions (i.e., "binding agent pharmaceutical compositions") that comprise and / or deliver one or more cells described herein, e.g., binding agents encoding or expressing a polypeptide described herein, e.g., anti-CD20 / anti-CD19 CARs. Binding agent pharmaceutical compositions are compositions that combine one or more cells described herein with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer such as neutral buffered saline, phosphate buffered saline, or the like, a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, or the like, a protein, polypeptide, or amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, an adjuvant (e.g., aluminum hydroxide), and a preservative.

[0254] The binding agent pharmaceutical composition of the present disclosure can be formulated for administration according to any of the embodiments set forth herein, at least one non-limiting example of which is intravenous administration. The composition may be formulated for intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and / or subcutaneous administration. Preferably, the composition is formulated for parenteral administration. Compositions suitable for parenteral administration may be aqueous or nonaqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes, for example, that render the composition isotonic with the blood of the intended recipient. Aqueous or nonaqueous sterile suspensions may 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 appropriate to the disease being treated (or prevented).

[0255] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using distilled water for injection as vehicle.For example, the isotonic solution containing physiological saline or glucose and other auxiliary agents such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be optionally combined with suitable solubilizers, for example, alcohols such as ethanol, polyalcohols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80 (trademark), HCO-50, and can be used as aqueous solutions for injection.

[0256] Non-limiting examples of oily liquids include sesame oil and soybean oil, which can be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other ingredients that can be included include a buffer such as phosphate buffer or sodium acetate buffer, a sedative such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.

[0257] In one embodiment, the binding agent pharmaceutical composition is substantially free of detectable levels of contaminants, such as endotoxin, mycoplasma, 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 media components, vector packaging cells or plasmid components, bacteria, and fungi. In one embodiment, the bacterium is selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus nucleus, and the like. The bacterial strain is at least one selected from the group consisting of Streptococcus pneumoniae, and / or Streptococcus pyogenes group A.

[0258] In various embodiments, the cells provided herein (e.g., binding agents, e.g., engineered T cells or engineered NK cells) may be activated and / or expanded from, and / or to produce, a binding agent pharmaceutical composition. In some embodiments, additional steps may be performed prior to administration to a subject. For example, a binding agent may be administered to a subject by transfecting immune cells with an inducible or constitutive expression construct described herein (e.g., an inducible or constitutive expression construct) prior to administration to the subject. The cells may be contacted (e.g., transduced or transfected) with an expression vector containing an expression construct and then expanded in vitro. In vitro expansion may continue for 1 or more days, e.g., 2 or more days, 3 or more days, 4 or more days, 6 or more days, or 8 or more days, before administration to a subject. In some embodiments, in vitro expansion may continue for 21 days or less, e.g., 18 days or less, 16 days or less, 14 days or less, 10 days or less, 7 days or less, or 5 days or less, before administration to a subject. For example, in vitro expansion may continue for 1 to 7 days, 2 to 10 days, 3 to 5 days, or 8 to 14 days, before administration to a subject. For example, a binding agent pharmaceutical composition comprising a binding agent (e.g., engineered T cells or engineered NK cells) may be administered in a desired dose, e.g., 10 days or less. 4 ~10 pieces 9 cells / kg body weight (e.g., 10 5 ~10 pieces 6Certain embodiments of the present disclosure include methods of administering to a subject a pharmaceutical composition described herein, such as a binding agent described herein (e.g., a population of engineered cells of the present disclosure), a protein therapeutic described herein, a composition comprising a binding agent, and / or a composition comprising a protein therapeutic, etc., in an amount effective to treat the subject, e.g., when administered in an appropriate dosing regimen.

[0259] In some embodiments, the binding agent is autologous to the subject, which can be immunologically naive, immunized, diseased, or in another state prior to isolating immune cells from the subject. In some embodiments, during in vitro expansion, the binding agent can be stimulated with an antigen (e.g., a TCR antigen). Antigen-stimulated expansion can optionally be supplemented with expansion under conditions that nonspecifically stimulate lymphocyte proliferation, such as, for example, anti-CD3 antibody, anti-Tac antibody, anti-CD28 antibody, or phytohemagglutinin (PHA). The expanded binding agent can be administered directly to the subject or frozen for future use, i.e., for later administration to the subject.

[0260] In some embodiments, the binding agent is treated ex vivo with interleukin-2 (IL-2) and then infused into the cancer patient, and the cancer patient is treated with IL-2 after infusion. Additionally, in some embodiments, the cancer patient can undergo preparatory lymphodepletion (temporary ablation of the immune system) prior to administration of the binding agent. The combination of IL-2 treatment and preparatory 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 temporally 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.

[0261] In certain embodiments, the binding agent is administered prior to, substantially simultaneously with, or after the administration of another therapeutic agent, such as a cancer therapeutic agent. The cancer therapeutic agent can be, for example, a chemotherapeutic agent, a biologic agent, or radiation therapy. In some embodiments, the subject receiving the binding agent is not administered a treatment sufficient to cause immune cell depletion, such as lymphodepleting chemotherapy or radiation therapy.

[0262] In some embodiments, the dosage administered to a subject may vary depending on the embodiment, the composition used, 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 a composition administered to a human or other subject to treat or prevent a condition. The exact amount of a composition required to be therapeutically effective may depend on many factors, such as the activity of the binding agent and the route of administration.

[0263] Any suitable number of binding agent cells may be administered to a subject. The synergist cells can proliferate to provide a therapeutic benefit, but in some embodiments, can be grown in a 10 2 More than one, e.g., 10 3 More than 10 pieces 4 More than 10 pieces 5 More than 10 8 In some embodiments, 10 or more binding agent cells are administered. 12 10 or less, for example 11 Less than or equal to 10 9 Less than or equal to 10 7 10 or less 5 No more than 10 binding agent cells described herein are administered to a subject. 2 ~10 pieces 5 pieces, 10 4 ~10 pieces 7 pieces, 10 3 ~10 pieces 9 Pieces or 10 5 ~10 pieces 10The binding agent pharmaceutical composition may be administered in an amount of, for example, 10 4 ~10 pieces 9 cells / kg body weight (e.g., 10 5 ~10 pieces 6 The binding agent pharmaceutical composition may be administered at a dose of, for example, about 2 x 10 cells / kg body weight. 6 cells / kg, approximately 3 x 10 6 cells / kg, approximately 4 x 10 6 cells / kg, approximately 5 x 10 6 cells / kg, approximately 6 x 10 6 cells / kg, approximately 7 x 10 6 cells / kg, approximately 8 x 10 6 cells / kg, approximately 9 x 10 6 cells / kg, approximately 1 x 10 7 cells / kg, approximately 2 x 10 7 cells / kg, approximately 3 x 10 7 cells / kg, approximately 4 x 10 7 cells / kg, approximately 5 x 10 7 cells / kg, approximately 6 x 10 7 cells / kg, approximately 7 x 10 7 cells / kg, approximately 8 x 10 7 cells / kg, or approximately 9 x 10 7 The cells may be administered at a dose of 100 cells / kg.

[0264] The dose of the binding agent described herein can be administered to a mammal at once or in a series of subdoses administered over a suitable period of time, for example, daily, semiweekly, weekly, biweekly, semimonthly, bimonthly, semi-annually, or yearly, as needed. A dosage unit containing an effective amount of the binding agent can be administered in a single daily administration, or the total daily dose can be administered in two, three, four, or more subdoses of the daily dose as needed.

[0265] The appropriate administration means can be selected by a physician. The administration route can be parenteral administration, for example, administration by injection, nasal administration, pulmonary administration, or transdermal administration. Administration can be systemic administration or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, the composition is selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The administration dose and administration method vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

[0266] In various embodiments, the binding agents described herein can be incorporated into pharmaceutical compositions. Pharmaceutical compositions containing the binding agents of the present disclosure can be formulated by known methods (see, e.g., Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985). In various examples, 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 physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Compositions comprising the binding agents of the present disclosure can include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0267] In various embodiments, compositions containing the binding agents described herein, e.g., sterile injectable preparations, can be formulated using distilled water for injection as a vehicle according to conventional pharmaceutical practice. For example, isotonic solutions containing saline or glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, optionally in combination with suitable solubilizers, e.g., alcohols such as ethanol and polyalcohols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as Polysorbate 80™ and HCO-50, can be used as injectable aqueous solutions. As disclosed herein, pharmaceutical compositions containing binding agents can exist in any form. Such forms include, for example, liquid forms (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, and semisolid forms. and solid dosage forms.

[0268] The selection or use of any form may depend, in part, on the intended mode of administration and therapeutic application. For example, compositions containing the binding agents of the present disclosure intended for systemic or local delivery may be in the form of an injectable or infusible solution. Thus, compositions containing the binding agents of the present disclosure may be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). Parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0269] The route of administration can be parenteral, e.g., by injection, intranasal, pulmonary, or transdermal. Administration can be systemic or local, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0270] In various embodiments, pharmaceutical compositions containing the binding agents of the present disclosure can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating a composition containing the binding agent of the present disclosure in the required amount in an appropriate solvent, optionally with one or a combination of the ingredients listed above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating a composition containing the binding agent of the present disclosure into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yield a powder of a composition containing the binding agent of the present disclosure and any additional desired ingredients (see below) from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of an injectable composition containing a binding agent of the present disclosure can be brought about by including in the composition containing the binding agent of the present disclosure an agent that delays absorption, for example, monostearate salts and gelatin.

[0271] Pharmaceutical compositions containing the binding agents of the present disclosure can be administered parenterally in the form of injectable formulations comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions containing the binding agents of the present disclosure can be administered parenterally in the form of injectable formulations comprising a therapeutic molecule and an injectable solution or suspension in sterile water and saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, etc. The formulations can be prepared by appropriately mixing the active ingredient with a pharmaceutically acceptable vehicle or medium, such as an excipient, vehicle, preservative, or binder, and then blending in a unit dosage form required by generally accepted pharmaceutical practice. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dosage within the specified range is provided. Non-limiting examples of oily liquids include sesame oil and soybean oil, which can be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other ingredients that can be included are buffers such as phosphate buffer or sodium acetate buffer, sedatives such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injections can be packaged in appropriate ampoules.

[0272] In some embodiments, compositions comprising a binding agent of the present disclosure may be formulated for storage at temperatures below 0° C. (e.g., −20° C. or −80° C.). In some embodiments, compositions comprising a binding agent of the present disclosure may be formulated for storage at 2° C. to 8° C. (e.g., 4° C.) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, some In embodiments, compositions comprising binding agents of the present disclosure are stable upon storage at 2°C to 8°C (e.g., 4°C) for at least 1 year.

[0273] In some cases, pharmaceutical compositions comprising the binding agents of the present disclosure can be formulated as a solution. In some embodiments, compositions comprising the binding agents of the present disclosure can be formulated as a buffered solution at an appropriate concentration, suitable for storage at, for example, 2°C to 8°C (e.g., 4°C). Pharmaceutical compositions comprising the binding agents described herein can be formulated into immunoliposome compositions. Liposomes with enhanced circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0274] In certain embodiments, compositions containing the binding agents of the present disclosure can be formulated with a carrier that will protect the composition from immediate release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for making such formulations are known. For example, see J. See R. Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0275] In some embodiments, compositions comprising a binding agent of the present disclosure may be formulated into a composition suitable for pulmonary 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 inhalation formulations and systems suitable for administering the formulations are also known. Pulmonary administration may be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, a composition comprising a binding agent of the present disclosure may 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, TURBOHALER™ (AstraZeneca; London, UK). Don), AIR™ inhaler (ALKERMES™; Cambridge, Massachusetts), ROTAHALER™ (GlaxoSmithKline; London, UK), and ECLIP SE™ (Sanofi-Aventis; Paris, France). See, e.g., 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 intrapulmonaryly via a metered-dose inhaler. These inhalers rely on propellants to deliver discrete doses of molecules to the lungs. Additional devices and methods of pulmonary administration are set forth, for example, in U.S. Patent Application Publication Nos. 2005 / 0271660 and 2009 / 0110679, the disclosures of each of which are incorporated herein by reference in their entireties.

[0276] In some embodiments, the composition comprising the binding agent 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 used for eye treatment can be in the form of a sterile aqueous solution, containing additional components such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants, stabilizers, non-ionic wetting or clarifying agents, and thickeners.The preparations described herein can be topically administered 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 eye drops or by immersing the eye in a treatment solution containing one or more compositions.

[0277] In certain embodiments, various devices for introducing drugs into the vitreous cavity of the eye may be suitable for administering compositions containing the binding agents of the present disclosure. For example, U.S. Patent Application Publication No. 2002 / 0026176 describes a device inserted through the sclera and protruding into the vitreous cavity. and describes a drug-containing plug capable of delivering a pharmaceutical agent into the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantable device for introduction into the suprachoroidal space or avascular region for sustained release of a drug 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 scleral surface of the eye. Additional methods and devices for delivering therapeutic agents to the eye (e.g., delivery via transscleral patches and 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, and 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 WO 04 / 073551, the disclosures of which are incorporated herein by reference in their entireties.

[0278] In various embodiments, subcutaneous administration may be achieved by a device such as a syringe, a pre-filled syringe, an autoinjector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, a portable syringe infusion pump with a subcutaneous infusion set, or other device for subcutaneous injection in combination with a binding agent drug.

[0279] The injection system of the present disclosure can use the delivery pen described in U.S. Pat. No. 5,308,341. Pen-type devices are commonly used for the self-delivery of insulin to patients with diabetes. Such devices can include at least one injection needle (e.g., a 31-gauge needle approximately 5 mm to 8 mm long) and are generally pre-filled with one or more therapeutic unit doses of a therapeutic solution, useful for quickly delivering the solution to a subject with minimal pain. One medication delivery pen includes a vial holder that can accommodate a vial of a therapeutic or other medication. The pen can be a completely mechanical device or can be combined with electronic circuitry to accurately set and / or indicate the dosage of medication to be injected to the user. See, e.g., U.S. Pat. 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 compositions featured in the present invention, including the binding agents of the present disclosure, are also described, for example, in U.S. Patent Nos. 6,277,099, 6,200,296, and 6,146,361 (the disclosures of each of which are incorporated herein by reference in their entirety). Microneedle-based pen devices are described, for example, in U.S. Patent No. 7,556,615 (the disclosure of which is incorporated herein by reference in its entirety). See also MOLLY™, a Precision Pen Injector (PPI) device manufactured by Scandinavian Health Ltd.

[0280] In some embodiments, compositions comprising binding agents of the present disclosure can be delivered to a subject by local administration that does not rely on transport of the binding agent via the vascular system to an intended target tissue or site. For example, compositions comprising binding agents of the present disclosure can be delivered by injection or implantation of a composition comprising a binding agent of the present disclosure, or by injection or implantation of a device containing a composition comprising a binding agent of the present disclosure. In certain embodiments, 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, can diffuse to an intended target tissue or site other than the site of administration.

[0281] In some embodiments, compositions comprising binding agents of the present disclosure may be administered locally to a joint, for example, directly to the joint (e.g., within the joint space) or near the joint. ) Examples of joints include the hip joint, knee joint, elbow joint, wrist joint, sternoclavicular joint, temporomandibular joint, and carpal joint. The disclosed compositions containing binding agents may also be administered to bursae, such as the acromion bursa, biceps radial bursa, cubitoradial bursa, deltoid bursa, infrapatellar bursa, ischial bursa, and any other bursa.

[0282] In some embodiments, the compositions comprising the binding agent of the present disclosure provided herein are in unit dosage forms suitable for self-administration.Such unit dosage forms can be provided in containers, generally, for example, vials, cartridges, pre-filled syringes or disposable pens.Dosage devices, such as those described in U.S. Patent No. 6,302,855, can also be used with the injection systems described herein.

[0283] A pharmaceutical solution can include a therapeutically effective amount of a composition comprising a binding agent of the present disclosure. Such an effective amount can be readily determined, in part, based on the effect of an administered composition comprising a binding agent of the present disclosure, or, when two or more agents are used, the combined effect of a composition comprising a binding agent of the present disclosure and one or more additional active agents. A therapeutically effective amount of a composition comprising a binding agent of the present disclosure can also vary according to factors such as the individual's medical condition, age, sex, and weight, and the ability of the composition (and one or more additional active agents) to cause a desired response in the individual, e.g., improvement of at least one condition parameter, e.g., improvement of at least one symptom of a complement-mediated disorder. For example, a composition comprising a therapeutically effective amount of a binding agent of the present disclosure can abrogate (reduce the severity or eliminate the occurrence of) and / or prevent the disorder and / or any one of the symptoms of the disorder. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or adverse effects of a composition comprising a binding agent of the present disclosure.

[0284] Compositions comprising binding agents of the present disclosure can be administered as a fixed dose or in milligram per kilogram (mg / kg) doses. In some embodiments, the dose can also be selected to reduce or avoid the production of antibodies or other host immune responses against one or more antigen-binding molecules in a composition comprising a binding agent of the present disclosure. While not intended to be limiting in any way, exemplary dosages of binding agents, such as compositions comprising a binding agent of the present disclosure, include, for example, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 100 mg / kg, 0.5 mg / kg to 50 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.5 mg / kg to 25 mg / kg, 1 mg / kg to 20 mg / kg, and 1 mg / kg to 10 mg / kg. Exemplary dosages of compositions comprising binding agents of the present disclosure include, but are not limited to, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg.

[0285] Suitable human doses of any of the compositions comprising the binding agents of the present disclosure can be further evaluated, for example, in Phase I dose escalation studies. See, e.g., 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.

[0286] In various embodiments, pharmaceutical compositions can include the nucleic acids, e.g., vectors, of the present disclosure. Methods for formulating pharmaceutical compositions containing the nucleic acids of the present disclosure are known, for example, from the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, an antibacterial agent such as benzyl alcohol or methylparaben, an antioxidant such as ascorbic acid or sodium bisulfite, a chelating agent such as ethylenediaminetetraacetic acid, an acid salt such as acetate, citrate, or phosphate. Buffers and agents for adjusting tonicity, such as sodium chloride or dextrose, may be included. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed, for example, in ampoules, disposable syringes, or two or more dose vials made of glass or plastic.

[0287] Pharmaceutical compositions containing the nucleic acids of the present disclosure suitable for injection may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0288] Sterility, stability, viscosity, and other factors related to effective therapeutic use can be considered. One way to maintain fluidity includes, for example, using a coating such as lecithin, maintaining a desired particle size, or using a surfactant. In some cases, it may be preferable to include an isotonic agent, such as a sugar, a polyalcohol such as mannitol or sorbitol, or sodium chloride in the composition. In some cases, prolonged absorption of an injectable composition containing the nucleic acid of the present disclosure can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin in the composition.

[0289] Sterile injectable solutions can be prepared by incorporating one or a combination of 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 a previously sterile-filtered solution thereof.

[0290] Oral compositions containing the nucleic acids of the present disclosure may contain an inert diluent or an edible carrier. For oral therapeutic administration, the nucleic acids of the present disclosure may be incorporated with excipients and used in the form of, for example, tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions containing the nucleic acids of the present disclosure may also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid, Primogel™, or corn starch; a lubricant such as magnesium stearate or Sterotes™; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavoring, or molecules of a similar nature.

[0291] In some embodiments, nucleic acids can be administered by any method suitable for administering nucleic acid agents, such as DNA vaccines. These methods include, for example, gene guns, bioinjectors, and skin patches, as well as needle-free methods, such as the microparticle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389 and the transdermal needle-free vaccination of mammals with powder-type vaccines disclosed in U.S. Pat. No. 6,168,587. Furthermore, intranasal delivery is possible, for example, as described in Hamajima et al. (1998) Clin. Immunol. Immunopathol. 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) can be used. In certain cases, microencapsulation can be used. Furthermore, biodegradable targets (e.g., as described in U.S. Pat. No. 6,471,996) can be used. Targetable microparticle delivery systems can be used.

[0292] Compositions containing nucleic acids of the present disclosure may contain components such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, mixtures thereof, or any other component included in a therapeutic composition containing nucleic acids. Nucleic acid compositions may contain, for example, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, phosphate-buffered saline solution, water, emulsions such as oil-in-water emulsions or water-in-oil emulsions, and various types of wetting agents compatible with pharmaceutical administration. Supplementary active molecules may also be incorporated into compositions containing nucleic acids of the present disclosure. Compositions containing nucleic acids of the present disclosure may contain stabilizers and preservatives, as well as any of the carriers described herein, with the further optional proviso that they are acceptable for in vivo use. For examples of additional carriers, stabilizers, and adjuvants, see Martin REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co.), Easton (1975) and See Williams & Williams, (1995), and "PHYSICIAN'S DESK REFERENCE," 52nd ed., Medical Economics, Montvale, NJ (1998).

[0293] The methods described herein include the preparation and use of pharmaceutical compositions comprising the nucleic acids of the present disclosure. Pharmaceutical compositions comprising the nucleic acids of the present disclosure are generally formulated to be compatible with their intended administration route. Examples of administration routes include parenteral administration, such as intravenous administration, intracranial administration, intradermal administration, subcutaneous administration, oral (e.g., inhalation) administration, transdermal (topical) administration, transmucosal administration, and rectal administration.

[0294] The nucleic acid composition may include a buffer or pH adjuster. The buffer may be a salt prepared from an organic acid or organic base. Buffers of the present disclosure include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, and phosphate buffers. Additional carriers include polyvinylpyrrolidone, Ficoll (a poly...

Claims

1. 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 sequences 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 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, and SEQ ID NO:199, and the sequences of three light chain CDRs (LCDRs) of the light chain variable region (LCVR) selected from the group consisting of 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, and SEQ ID NO:

210.

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

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

4. The antigen binding system, antibody, or antigen binding fragment thereof, comprises a first domain comprising three heavy chain complementarity determining regions (HCDRs) and a second domain comprising three light chain complementarity determining regions (LCDRs), wherein: The HCDR and the LCDR are (i) HCDR1 according to any one of SEQ ID NOs: 3 to 5, HCDR2 according to any one of SEQ ID NOs: 6 to 8, HCDR3 according to any one of SEQ ID NOs: 9 to 11, LCDR1 according to any one of SEQ ID NOs: 14 to 16, LCDR2 according to any one of SEQ ID NOs: 17 to 19, LCDR3 according to any one of SEQ ID NOs: 20 to 22, (ii) HCDR1 according to any one of SEQ ID NOs: 25 to 27, HCDR2 according to any one of SEQ ID NOs: 28 to 30, HCDR3 according to any one of SEQ ID NOs: 31 to 33, LCDR1 according to any one of SEQ ID NOs: 36 to 38, LCDR2 according to any one of SEQ ID NOs: 39 to 41, LCDR3 according to any one of SEQ ID NOs: 42 to 44, (iii) HCDR1 according to any one of SEQ ID NOs: 47 to 49, HCDR2 according to any one of SEQ ID NOs: 50 to 52, HCDR3 according to any one of SEQ ID NOs: 53 to 55, LCDR1 according to any one of SEQ ID NOs: 58 to 60, LCDR2 according to any one of SEQ ID NOs: 61 to 63, LCDR3 according to any one of SEQ ID NOs: 64 to 66; (iv) HCDR1 according to any one of SEQ ID NOs: 69 to 71, HCDR2 according to any one of SEQ ID NOs: 72 to 74, HCDR3 according to any one of SEQ ID NOs: 75 to 77, LCDR1 according to any one of SEQ ID NOs: 80 to 82, LCDR2 according to any one of SEQ ID NOs: 83 to 85, LCDR3 according to any one of SEQ ID NOs: 86 to 88; (v) an HCDR1 according to any one of SEQ ID NOs: 91 to 93, an HCDR2 according to any one of SEQ ID NOs: 94 to 96, an HCDR3 according to any one of SEQ ID NOs: 97 to 99, an LCDR1 according to any one of SEQ ID NOs: 102 to 104, an LCDR2 according to any one of SEQ ID NOs: 105 to 107, and an LCDR3 according to any one of SEQ ID NOs: 108 to 110; (vi) HCDR1 according to any one of SEQ ID NOs: 113 to 115, HCDR2 according to any one of SEQ ID NOs: 116 to 118, HCDR3 according to any one of SEQ ID NOs: 119 to 121, LCDR1 according to any one of SEQ ID NOs: 124 to 126, LCDR2 according to any one of SEQ ID NOs: 127 to 129, LCDR3 according to any one of SEQ ID NOs: 130 to 132; (vii) HCDR1 according to any one of SEQ ID NOs: 135 to 137, HCDR2 according to any one of SEQ ID NOs: 138 to 140, HCDR3 according to any one of SEQ ID NOs: 141 to 143, and LCD according to any one of SEQ ID NOs: 146 to 148. R1, LCDR2 according to any one of SEQ ID NOs: 149 to 151, LCDR3 according to any one of SEQ ID NOs: 152 to 154, (viii) an HCDR1 according to any one of SEQ ID NOs: 157 to 159, an HCDR2 according to any one of SEQ ID NOs: 160 to 162, an HCDR3 according to any one of SEQ ID NOs: 163 to 165, an LCDR1 according to any one of SEQ ID NOs: 168 to 170, an LCDR2 according to any one of SEQ ID NOs: 171 to 173, and an LCDR3 according to any one of SEQ ID NOs: 174 to 176; (ix) an HCDR1 according to any one of SEQ ID NOs: 179 to 181, an HCDR2 according to any one of SEQ ID NOs: 182 to 184, an HCDR3 according to any one of SEQ ID NOs: 185 to 187, an LCDR1 according to any one of SEQ ID NOs: 190 to 192, an LCDR2 according to any one of SEQ ID NOs: 193 to 195, an LCDR3 according to any one of SEQ ID NOs: 196 to 198, or (x) an HCDR1 according to any one of SEQ ID NOs: 201 to 203, an HCDR2 according to any one of SEQ ID NOs: 204 to 206, an HCDR3 according to any one of SEQ ID NOs: 207 to 209, an LCDR1 according to any one of SEQ ID NOs: 212 to 214, an LCDR2 according to any one of SEQ ID NOs: 215 to 217, and an LCDR3 according to any one of SEQ ID NOs: 218 to 220; Including, An antigen-binding system, antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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

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

210.

6. An antigen-binding system, antibody, or antigen-binding fragment thereof according to claim 5.

7. 7. The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 1, wherein the three HCDRs and the three LCDRs are comprised in a single polypeptide.

8. 7. The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 1, wherein the three HCDRs are comprised in a first polypeptide and the three LCDRs are comprised in a second polypeptide.

9. 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, (i) 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 a binding motif that specifically binds to an antigen selected from the group consisting of HER2 / Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, IL-11Rα, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-1, mutant p53, mutant ras, prostate-specific antigen, ROR1, VEGFR2, EphA3 (EPH receptor A3), BAFFR (B-cell activating factor receptor), and combinations thereof; and / or (ii) a binding motif that specifically binds to a B-cell characteristic antigen (wherein, optionally, the B-cell characteristic antigen is not CD19 or CD20); Further comprising: An antigen-binding system, antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The antigen-binding system, antibody or antigen-binding fragment thereof according to 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 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 include HCDR1, HCDR2, and HCDR3; the three LCDRs of the anti-CD19 binding motif include LCDR1, LCDR2, and LCDR3; The HCDR and the LCDR of the anti-CD19 binding motif are: HCDR1 according to any one of SEQ ID NOs: 223 to 225; HCDR2 according to any one of SEQ ID NOs: 226 to 228; HCDR3 according to any one of SEQ ID NOs: 229 to 231; LCDR1 according to any one of SEQ ID NOs: 234 to 236; 239, an LCDR2 according to any one of SEQ ID NOs: 240 to 242, 12. An antigen-binding system, antibody, or antigen-binding fragment thereof according to claim 11.

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

15. 15. The antigen-binding system, antibody, or antigen-binding fragment thereof of any one of claims 12 to 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 all comprised in 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 in a chimeric antigen receptor.

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

18. 15. 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 both comprised in a second, different polypeptide.

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

20. 20. The antigen-binding system, antibody, or antigen-binding fragment thereof of claim 18 or 19, wherein the second polypeptide is or is comprised in 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. 23. A method of making an engineered cell, comprising transfecting or transducing a cell with the nucleic acid of claim 21 or the vector of claim 22.

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

25. 21. A method of treating cancer in a subject in need thereof, 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 to 20.

26. 21. A method of eliciting an immune response in a subject or immunizing a subject against cancer, 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 to 20.

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

28. The cancers include acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelomatous leukemia (CML), chronic myelogenous 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 syndromes, 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 dysplasmocytosis, 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 28. The method of any one of claims 25-27, wherein the tumor is selected from the group consisting of idiopathies, T-cell acute lymphoblastic 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 to 28, wherein the cell therapy is an allogeneic cell therapy or an autologous cell therapy.

Images