CD19-targeted chimeric antigen receptors and uses thereof

Abstract

The presently disclosed subject matter provides a chimeric antigen receptor (CAR) that specifically targets CD19 and cells comprising such a CD19-targeted CAR. The presently disclosed subject matter further provides the use of a CD19-targeted CAR for treatment, for example, for treating blood cancer. In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds to CD19, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv), Fab, or F(ab)2. In certain embodiments, the extracellular antigen-binding domain comprises an scFv. In certain embodiments, the scFv is a human scFv.

Description

[Technical Field]

[0001] 1. Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 015,362, filed April 24, 2020, and U.S. Provisional Patent Application No. 63 / 073,133, filed September 1, 2020, the contents of each of which are incorporated by reference in their entirety and from which priority is claimed.

[0002] 2.Technical Field The presently disclosed subject matter provides methods for treating neoplasms (e.g., cancer) using cells comprising a chimeric antigen receptor (CAR) that specifically targets CD19. [Background technology]

[0003] 3.Background Cell-based immunotherapy is a potentially curative treatment for cancer. T cells and other immune cells can be engineered to target tumor antigens by introducing genetic material encoding an artificial or synthetic receptor for the antigen, called a chimeric antigen receptor (CAR), specific for the selected antigen. Recent clinical success has been shown for the treatment of hematological malignancies with targeted T cell therapy using CARs.

[0004] It has been shown that T cells expressing CD19-specific CAR with binding domain derived from mouse monoclonal antibody can treat B cell malignant lesions.In human patients, the T cell-mediated immune response specific to the mouse scFv antigen-binding domain of CAR occurs, which may result in the early elimination of CAR T cells, thereby increasing the risk of tumor recurrence.Therefore, there is a need for an improved CD19-targeted CAR with reduced toxicity and immunogenicity and / or improved safety and efficacy. Summary of the Invention [Means for solving the problem]

[0005] 4. Summary of the Invention The presently disclosed subject matter provides CD19-targeted chimeric antigen receptors (CARs), cells comprising CD19-targeted CARs, and uses of the cells for treatment, e.g., to treat neoplasms.

[0006] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds to CD19, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv), Fab, or F(ab)2. In certain embodiments, the extracellular antigen-binding domain comprises an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the extracellular antigen-binding domain comprises: (a) a heavy chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9 or a conservative modification thereof; and / or (b) i) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof; ii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof; iii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 or a conservative modification thereof; iv) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof; v) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof; vi) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof; vii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof; or viii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof. The light chain variable region comprises:

[0007] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9.

[0008] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0009] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0010] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0011] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57.

[0012] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59.

[0013] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0014] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63.

[0015] In certain embodiments, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0016] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0017] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0018] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0019] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57.

[0020] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59.

[0021] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0022] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63.

[0023] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0024] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0025] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, or SEQ ID NO: 22.

[0026] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 8, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14, SEQ ID NO:18, or SEQ ID NO:22.

[0027] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:14.

[0028] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:18.

[0029] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:22.

[0030] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:58.

[0031] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:60.

[0032] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:62.

[0033] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:64.

[0034] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:67.

[0035] In certain embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, the heavy chain variable region and the light chain variable region are, from the N-terminus to the C-terminus, L -V H This is its position.

[0036] In certain embodiments, the extracellular antigen-binding domain comprises or is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 23.

[0037] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide.

[0038] In certain embodiments, the intracellular signaling domain comprises a CD3ζ polypeptide. In certain embodiments, the CD3ζ polypeptide is a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 consisting of two loss-of-function mutations. In certain embodiments, the native ITAM1 consists of the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the ITAM2 variant consists of the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the ITAM3 variant consists of the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43.

[0039] In certain embodiments, the intracellular signaling domain further comprises at least one costimulatory signaling region. In certain embodiments, at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. In certain embodiments, at least one costimulatory signaling region comprises the intracellular domain of CD28 or a portion thereof, the intracellular domain of 4-1BB or a portion thereof, the intracellular domain of OX40 or a portion thereof, the intracellular domain of ICOS or a portion thereof, or the intracellular domain of DAP-10 or a portion thereof. In certain embodiments, at least one costimulatory signaling region comprises a CD28 polypeptide.

[0040] In certain embodiments, the CAR is expressed from a vector, hi certain embodiments, the vector is a retroviral vector.

[0041] The presently disclosed subject matter further provides a cell comprising a CAR disclosed herein. In certain embodiments, the cell is transduced with the CAR. In certain embodiments, the CAR is constitutively expressed on the surface of the cell. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a lymphoid or myeloid lineage cell. In certain embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a stem cell capable of differentiating into a lymphoid cell, and a stem cell capable of differentiating into a myeloid cell. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a tumor-infiltrating lymphocyte (TIL), a natural killer T cell, a mucosal-associated invariant T cell, and a gamma delta T cell. In certain embodiments, the cell is an NK cell. In certain embodiments, the NK cell is derived from a stem cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cells are embryoid stem cells or induced pluripotent stem cells.

[0042] Furthermore, the presently disclosed subject matter provides a nucleic acid molecule encoding a CAR disclosed herein. In certain embodiments, the nucleic acid molecule further comprises a promoter operably linked to the CAR. The promoter may be endogenous or exogenous. In certain embodiments, the promoter is an exogenous promoter. In certain embodiments, the exogenous promoter is selected from the group consisting of an elongation factor (EF)-1 promoter, a cytomegalovirus immediate early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter, and a ubiquitin C promoter. In certain embodiments, the promoter is an endogenous promoter. In certain embodiments, the endogenous promoter is selected from a TCR alpha promoter, a TCR beta promoter, and a beta2-microglobulin promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a 4-1BB promoter, a PD1 promoter, and a LAG3 promoter.

[0043] The presently disclosed subject matter also provides vectors comprising the nucleic acid molecules disclosed herein. In certain embodiments, the vector is a retroviral vector.

[0044] The presently disclosed subject matter further provides a cell expressing a nucleic acid molecule disclosed herein. In certain embodiments, the cell is a T cell or a natural killer (NK) cell.

[0045] The subject matter of the present disclosure provides a composition comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In certain embodiments, the composition comprises about 1 x 10 6 From about 5 x 10 8In certain embodiments, the composition comprises between about 1 x 10 cells. 6 From about 1 x 10 8 In certain embodiments, the composition comprises between about 1 x 10 cells. 6 From about 5 x 10 7 In certain embodiments, the composition comprises between about 2.5 x 10 cells. 7 Contains cells.

[0046] The presently disclosed subject matter further provides various methods for using the cells of the present disclosure. The presently disclosed subject matter provides a method for reducing tumor burden in a subject. In certain embodiments, the method comprises administering to a subject the cells or compositions disclosed herein. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in a subject.

[0047] The presently disclosed subject matter provides methods for increasing or prolonging survival of a subject having a neoplasm. In certain embodiments, the method comprises administering to the subject a cell or composition disclosed herein.

[0048] The presently disclosed subject matter provides methods for treating and / or preventing a neoplasm in a subject. In certain embodiments, the method comprises administering to the subject a cell or composition disclosed herein.

[0049] The cells and compositions of the present disclosure can be used for treatment.In certain embodiments, the cells and compositions of the present disclosure are used to treat and / or prevent neoplasms in subjects.In certain embodiments, the cells and compositions of the present disclosure are used to increase or prolong the survival of subjects with neoplasms.In certain embodiments, the cells and compositions of the present disclosure are used to treat and / or prevent neoplasms in subjects.

[0050] In certain embodiments, the tumor and / or neoplasm is CD19-associated. In certain embodiments, the tumor and / or neoplasm is a hematological cancer. In certain embodiments, the hematological cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma. In certain embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the tumor and / or neoplasm is a B-cell malignancy. In certain embodiments, the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma. In certain embodiments, the tumor and / or neoplasm is a B-cell lymphoma. In certain embodiments, the B-cell lymphoma is a relapsed or refractory (R / R) B-cell lymphoma. In certain embodiments, the subject is a human subject.

[0051] Furthermore, the presently disclosed subject matter provides methods for producing cells comprising a CAR disclosed herein. In certain embodiments, the method comprises introducing into a cell a nucleic acid molecule encoding a CAR disclosed herein.

[0052] Furthermore, the presently disclosed subject matter provides kits for reducing tumor burden in a subject, treating and / or preventing a neoplasm in a subject, and / or increasing or prolonging survival of a subject with a neoplasm. In certain embodiments, the kits include the cells disclosed herein. In certain embodiments, the kits further include instructions for using the cells to reduce tumor burden in a subject, treating and / or preventing a neoplasm in a subject, and / or increasing or prolonging survival of a subject with a neoplasm. In certain embodiments, for example, the following items are provided: (Item 1) A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that specifically binds to CD19, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is a) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof; and / or b) i) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof; ii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof; iii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 or a conservative modification thereof; iv) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof; v) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof; vi) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof; vii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof; or viii) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof. a light chain variable region comprising Including, CAR. (Item 2) The extracellular antigen-binding domain is a single-chain variable fragment (scFv), Fab, or F(ab). 2 2. The CAR of item 1, comprising: (Item 3) 3. The CAR of item 2, wherein the extracellular antigen-binding domain comprises an scFv. (Item 4) 4. The CAR of item 3, wherein the scFv is a human scFv. (Item 5) 5. The CAR of any one of items 1 to 4, wherein the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. (Item 6) the light chain variable region (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 (d) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57; (e) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59; (f) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61; (g) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63; or (h) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66 6. The CAR of any one of items 1 to 5, comprising: (Item 7) the light chain variable region (a) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 7. The CAR of any one of items 1 to 6, comprising: (Item 8) (a) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13; (b) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:17; (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21; (d) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:57; (e) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:59; (f) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61; (g) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:63; or (h) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66; 8. The CAR according to any one of items 1 to 7. (Item 9) (a) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13; (b) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:17; or (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21; 9. The CAR according to any one of items 1 to 8. (Item 10) 10. The CAR of any one of items 1 to 9, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 10. (Item 11) 11. The CAR of any one of items 1 to 10, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10. (Item 12) 12. The CAR of any one of items 1 to 11, wherein the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67. (Item 13) 13. The CAR of any one of items 1 to 12, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67. (Item 14) 14. The CAR of any one of items 1 to 13, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, or SEQ ID NO: 22. (Item 15) (b) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 10; and 8. The CAR of any one of items 1 to 14, comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67. (Item 16) 16. The CAR of any one of items 1 to 15, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67. (Item 17) 17. The CAR of any one of items 1 to 16, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, or SEQ ID NO: 22. (Item 18) (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18; (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 22; (d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58; (e) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60; (f) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62; (g) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64; or (h) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67; 18. The CAR according to any one of items 1 to 17. (Item 19) (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18; or (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 22; 19. The CAR according to any one of items 1 to 18. (Item 21) 21. The CAR of any one of items 1 to 20, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region. (Item 22) 22. The CAR of item 21, wherein the linker consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. (Item 23) The heavy chain variable region and the light chain variable region are, from N-terminus to C-terminus, V L -V H 23. The CAR of any one of items 1 to 22, wherein the CAR is positioned at (Item 24) 24. The CAR of any one of items 1 to 23, wherein the extracellular antigen-binding domain comprises an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 23, or is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 23. (Item 25) 25. The CAR of any one of items 1 to 24, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. (Item 26) 26. The CAR of item 25, wherein the transmembrane domain comprises a CD28 polypeptide. (Item 27) 27. The CAR of any one of items 1 to 26, wherein the intracellular signaling domain comprises a CD3ζ polypeptide. (Item 28) The CAR of item 27, wherein the CD3ζ polypeptide is a modified CD3ζ polypeptide. (Item 29) 29. The CAR of item 28, wherein the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 consisting of two loss-of-function mutations. (Item 30) 30. The CAR of item 29, wherein the native ITAM1 consists of the amino acid sequence set forth in SEQ ID NO: 31. (Item 31) 30. The CAR of item 29, wherein the ITAM2 variant consists of the amino acid sequence set forth in SEQ ID NO: 37. (Item 32) 30. The CAR of item 29, wherein the ITAM3 variant consists of the amino acid sequence set forth in SEQ ID NO: 41. (Item 33) 33. The CAR of any one of items 28 to 32, wherein the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43. (Item 34) 34. The CAR of any one of paragraphs 1 to 33, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region. (Item 35) 35. The CAR of item 34, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. (Item 36) 36. The CAR of paragraph 35, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide. (Item 37) 37. The CAR of any one of items 1 to 36, which is expressed from a vector. (Item 38) 38. The CAR of item 37, wherein the vector is a retroviral vector. (Item 39) 39. A cell comprising the CAR of any one of items 1 to 38. (Item 40) 40. The cell of item 39, wherein the CAR is transduced. (Item 41) 41. The cell of claim 39 or 40, wherein the CAR is constitutively expressed on the surface of the cell. (Item 42) 42. The cell of any one of items 39 to 41, which is an immunoresponsive cell. (Item 43) 43. The cell of any one of items 39 to 42, which is a cell of lymphoid lineage or a cell of myeloid lineage. (Item 44) 44. The cell of any one of items 39 to 43, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a stem cell capable of differentiating into a lymphoid cell, and a stem cell capable of differentiating into a myeloid cell. (Item 45) 45. The cell of any one of items 39 to 44, which is a T cell. (Item 46) 46. ​​The cell of claim 38 or 45, wherein the T cell is selected from the group consisting of helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. (Item 47) 45. The cell of any one of items 39 to 44, which is a NK cell. (Item 48) Item 48. The cell of item 47, wherein the NK cell is derived from a stem cell. (Item 49) 49. The cell of item 44 or 48, wherein the stem cell is a pluripotent stem cell. (Item 50) 50. The cell of item 49, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. (Item 51) 39. A nucleic acid molecule encoding the CAR of any one of items 1 to 38. (Item 52) 52. The nucleic acid molecule of claim 51, further comprising a promoter operably linked to the CAR. (Item 53) 53. The nucleic acid molecule of item 52, wherein the promoter is endogenous or exogenous. (Item 54) 54. The nucleic acid molecule of item 53, wherein the exogenous promoter is selected from the group consisting of an elongation factor (EF)-1 promoter, a cytomegalovirus immediate early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter, and a ubiquitin C promoter. (Item 55) 52. The nucleic acid molecule of item 51, wherein the promoter is an inducible promoter. (Item 56) 56. The nucleic acid molecule of item 55, wherein the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a 4-1BB promoter, a PD1 promoter, and a LAG3 promoter. (Item 57) 54. The nucleic acid molecule of item 53, wherein the promoter is an endogenous promoter. (Item 58) 58. The nucleic acid molecule of item 57, wherein the endogenous promoter is selected from a TCR alpha promoter, a TCR beta promoter, and a beta2-microglobulin promoter. (Item 59) 59. A vector comprising the nucleic acid molecule of any one of items 51 to 58. (Item 60) 60. The vector according to item 59, which is a retroviral vector. (Item 61) 59. A cell expressing the nucleic acid molecule of any one of items 51 to 58. (Item 62) 62. The cell of item 61, which is a T cell or a natural killer (NK) cell. (Item 63) A composition comprising the cells of any one of items 39 to 50, 61 and 62. (Item 64) 64. The composition according to item 63, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. (Item 65) Approximately 1×10 6 From about 5 x 10 8 65. The composition of claim 63 or 64, comprising cells between 65 and 65. (Item 66) Approximately 1×106 From about 1 x 10 8 65. The composition of claim 63 or 64, comprising cells between 65 and 65. (Item 67) Approximately 1×10 6 From about 5 x 10 7 65. The composition of claim 63 or 64, comprising cells between 65 and 65. (Item 68) Approximately 2.5×10 7 68. The composition of any one of items 63 to 67, comprising cells. (Item 69) 62. A method of reducing tumor burden in a subject, comprising administering to the subject a cell of any one of items 39 to 50, 61 and 62 or a composition of any one of items 63 to 68. (Item 70) 70. The method of claim 69, wherein the method reduces the number of tumor cells, shrinks tumor size, and / or eradicates tumors in the subject. (Item 71) 62. A method for increasing or prolonging survival of a subject having a neoplasm, comprising administering to the subject a cell described in any one of items 39 to 50, 61 and 62, or a composition described in any one of items 63 to 68. (Item 72) 62. A method for treating and / or preventing a neoplasm in a subject, comprising administering to the subject a cell described in any one of items 39 to 50, 61 and 62 or a composition described in any one of items 63 to 68. (Item 73) 73. The method of any one of items 69 to 72, wherein the tumor and / or neoplasm is CD19 associated. (Item 74) 74. The method of any one of items 69 to 73, wherein the tumor and / or neoplasm is a hematological cancer. (Item 75) 75. The method of claim 74, wherein the hematological cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma. (Item 76) 76. The method of item 75, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. (Item 77) 76. The method of claim 75, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. (Item 78) 74. The method of any one of items 69 to 73, wherein the tumor and / or neoplasm is a B-cell malignancy. (Item 79) 79. The method of item 78, wherein the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma. (Item 80) 80. The method of any one of items 69 to 79, wherein the tumor and / or neoplasm is a B-cell lymphoma. (Item 81) 81. The method of item 80, wherein the B-cell lymphoma is relapsed or refractory (R / R) B-cell lymphoma. (Item 82) 82. The method of any one of items 69 to 81, wherein the subject is a human subject. (Item 83) 69. The cell of any one of items 39 to 50, item 61 and item 62 or the composition of any one of items 63 to 68 for use in therapy. (Item 84) 64. The cell of any one of items 39 to 50, item 61 and item 62 or the composition of any one of items 63 to 68 for use in the treatment and / or prevention of a neoplasm in a subject. (Item 85) The cell of any one of items 39 to 50, item 61 and item 62 or the composition of any one of items 63 to 68 for use in increasing or prolonging the survival of a subject having a neoplasm. (Item 86) 64. The cell of any one of items 39 to 50, item 61 and item 62 or the composition of any one of items 63 to 68 for use in the treatment and / or prevention of a neoplasm in a subject. (Item 87) 87. The cell or composition for use according to any one of items 84 to 86, wherein the tumor and / or neoplasm is CD19 associated. (Item 88) 88. The cell or composition for use according to any one of items 84 to 87, wherein the tumor and / or neoplasm is a blood cancer. (Item 89) 89. The cell or composition for use according to item 88, wherein the hematological cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma. (Item 90) 90. The cell or composition for use of item 89, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. (Item 91) 90. The cell or composition for use according to item 89, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. (Item 92) 88. The cell or composition for use according to any one of items 84 to 87, wherein the tumor and / or neoplasm is a B-cell malignancy. (Item 93) 93. The cell or composition for use of item 92, wherein the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma. (Item 94) 94. The cell or composition for use according to any one of items 84 to 93, wherein the tumor and / or neoplasm is a B-cell lymphoma. (Item 95) 95. The cell or composition for use according to item 94, wherein the B-cell lymphoma is relapsed or refractory (R / R) B-cell lymphoma. (Item 96) 96. The cell or composition for use according to any one of items 84 to 95, wherein the subject is a human subject. (Item 97) 40. A method for generating a cell comprising the CAR of any one of items 1 to 38, the method comprising introducing into the cell a nucleic acid molecule encoding the CAR of any one of items 1 to 38. (Item 98) 63. A kit for reducing tumor burden in a subject, for treating and / or preventing a neoplasm in a subject, and / or for increasing or prolonging survival of a subject having a neoplasm, the kit comprising the cell of any one of items 39 to 50, 61, and 62. (Item 99) 99. The kit of item 98, further comprising instructions for using the cells to reduce tumor burden in a subject, to treat and / or prevent a neoplasm in a subject, and / or to increase or prolong survival of a subject having a neoplasm. 5. Brief description of the drawings [Brief explanation of the drawings]

[0053] [Figure 1]Figures 1A and 1B show the cytotoxicity of T cells containing a CD19-targeted CAR against cells expressing CD19. Figure 1A shows killing of CD19-expressing Raji cancer cell lines. Figure 1B shows killing of CD19-knockout Raji cells. The positive control is 1928z-1XX CAR.

[0054] [Figure 2] Figures 2A and 2B show the cytotoxicity of T cells containing a CD19-targeted CAR against cells expressing CD19, as measured by flow-based killing assay. Figure 2A shows killing against a CD19-expressing Raji cancer cell line. Figure 2B shows killing against CD19 knockout Raji cells. The positive control is the 1928z-1XX CAR.

[0055] [Figure 3] Figures 3A and 3B show IL-2 cytokine secretion by T cells containing a CD19-targeted CAR. Figure 3A shows IL-2 cytokine secretion by T cells containing a CD19-targeted CAR when co-cultured with a CD19-expressing Raji cancer cell line. Figure 3B shows IL-2 cytokine secretion by T cells containing a CD19-targeted CAR when co-cultured with CD19-knockout Raji cells. The positive control is 1928z-1XX CAR.

[0056] [Figure 4] Figures 4A and 4B show IFN-γ cytokine secretion by T cells containing a CD19-targeted CAR. Figure 4A shows IFN-γ cytokine secretion by T cells containing a CD19-targeted CAR when co-cultured with a CD19-expressing Raji cancer cell line. Figure 4B shows IFN-γ cytokine secretion by T cells containing a CD19-targeted CAR when co-cultured with CD19-knockout Raji cells. The positive control is 1928z-1XX CAR.

[0057] [Figure 5]Figure 5 shows the in vivo anti-tumor activity of T cells containing a CD19-targeted CAR. #17 is a negative control, a CD19-targeted CAR with no detectable in vivo activity.

[0058] [Figure 6] FIG. 6 shows the amino acid sequence of CD19-HSA-His10.

[0059] [Figure 7] Figure 7 shows binding data for #2 scFv, SJ25c1 scFv, and FMC63 scFv. Relative competition for a common epitope within soluble CD19 antigen was assessed using a Biacore surface plasmon resonance (SPR) assay. A mixture of soluble CD19-specific antibody (FMC63 or SJ25c1) and CD19 antigen was flowed over a chip containing #2 scFv to determine whether #2 scFv cross-competes with the same epitope as SJ25c1 scFv and FMC63 scFv. 19(T2) represents "#2 scFv."

[0060] [Figure 8] Figure 8 shows a gamma-retroviral vector containing the #2 CAR. "19(T2)" stands for "#2." The #2 CAR contains a CD19-specific T2 (19(T2)) scFv (or "#2 scFv"), a CD8 alpha leader peptide, a CD28 gene fragment, and a modified CD3ζ intracellular signaling domain. The CD28 gene fragment includes its extracellular hinge, transmembrane, and intracellular domains. The modified CD3ζ chain has one functional wild-type and two mutated immunoreceptor tyrosine-based activation motifs (ITAMs), designated "1XX." The amino acid sequence of the modified CD3ζ intracellular signaling domain is set forth in SEQ ID NO: 43. An alignment of the amino acid sequences of CD3ζ-1XX and CD3ζ-wild-type is shown in Figure 9. Amino acid differences between the two sequences are marked.

[0061] [Figure 9]shows an alignment of the amino acid sequences of CD3ζ-1XX and CD3ζ-wild type.

[0062] [Figure 10] Figure 10 shows the in vitro cytotoxic activity of #2 CAR-T cells and 1928z-1XX CAR T cells. "19(T2)28z1xx" represents "#2 CAR."

[0063] [Figure 11] Figure 11 shows tumor elimination by #2 CAR T cells and 1928z-1XX CAR T cells. "19(T2)28z1xx" represents "#2 CAR."

[0064] [Figure 12] Figure 12 shows a gamma-retroviral vector containing the 1928z CAR. The amino acid sequence of wild-type hCD3ζ is shown in Figure 9 as SEQ ID NO:30.

[0065] [Figure 13] Figure 13 shows CAR expression in gamma-retrovirally transduced CD4+ and CD8+ T cells. "19(T2)28z1XX" represents "#2 CAR."

[0066] [Figure 14-1] Figure 14 shows the phenotypes of 1928z CAR T cells and #2 CAR T cells. "19(T2)28z1XX" represents "#2 CAR." [Figure 14-2] Figure 14 shows the phenotypes of 1928z CAR T cells and #2 CAR T cells. "19(T2)28z1XX" represents "#2 CAR."

[0067] [Figure 15] Figure 15 shows the kinetics of tumor eradication by #2 CAR T cells and 1928z CAR T cells. "19(T2)28z1XX" represents "#2 CAR." Each curve represents the tumor burden in one mouse.

[0068] [Figure 16] Figure 16 shows survival of NALM6 leukemia-bearing mice after iv infusion of #2 CAR T cells or 1928z CAR T cells. "19(T2)28z1XX" represents "#2 CAR."

[0069] [Figure 17] Figure 17 shows CAR T cell quantification and phenotyping in the bone marrow of mice treated with #2 CAR T cells or 1928z CAR T cells (day 17). All data are mean ± sem, and p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0070] [Figure 18] Figure 18 shows the body weight of mice after treatment with #2 CAR T cells or 1928z CAR T cells. "19(T2)28z1XX" represents "#2 CAR." Asterisks indicate the time point at which unscheduled death occurred. Short curves represent the death of all mice at the indicated time point.

[0071] [Figure 19] Figure 19 shows quantification of liver enzymes in serum at 10 and 27 / 28 days post-infusion in mice treated with 1x106 CAR T cells. Statistical comparisons were performed on the same days between the two cohorts. All data are mean + sem; p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0072] [Figure 20]Figure 20 shows quantification of human cytokine levels at 10 and 27 / 28 days post-infusion in mice treated with 1x106 #2 CAR T cells or 1928z CAR T cells. All data are mean + sem; p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0073] [Figure 21] Quantification of mouse cytokine levels at 10 days and 27 / 28 days post-infusion in mice treated with 1x106 #2 CAR T cells or 1928z CAR T cells. All data are mean ± sem; p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0074] [Figure 22] Figure 22 shows the quantification of essential blood cells at 27 / 28 days post-infusion in mice treated with #2 CAR T cells and 1928z CAR T cells. All data are mean + sem; p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0075] [Figure 23] Figure 23 shows a comparison of body weight and vital organ weights at 27 / 28 days post-infusion for mice treated with #2 CAR T cells and 1928z CAR T cells. The number of mice per group is indicated in the graph legend. All data are mean + sem; p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0076] [Figure 24]Figure 24 shows quantification of lymphocytes and tumor infiltration in the liver, spleen, and bone marrow of mice treated with CAR T cells. All data are mean ± sem; p values ​​were calculated using a two-tailed unpaired Student's t-test. "19(T2)28z1XX" represents "#2 CAR."

[0077] [Figure 25] FIG. 25 shows exemplary flow cytometry chromatograms of anti-CD19 antibodies binding to CD19-expressing wild-type Raji and NALM-6 cells and CD19 knockout Raji and NALM-6 cells.

[0078] [Figure 26-1] 26A-26C show exemplary binding curves of anti-CD19 antibodies binding to CD19 on NALM-6 cells. [Figure 26-2] 26A-26C show exemplary binding curves of anti-CD19 antibodies binding to CD19 on NALM-6 cells. DETAILED DESCRIPTION OF THE INVENTION

[0079] 6. Detailed Description The presently disclosed subject matter provides a chimeric antigen receptor (CAR) that specifically targets CD19 and cells comprising such a CD19-targeted CAR. The cells can be immunoresponsive cells, for example, genetically modified immunoresponsive cells (e.g., T cells or NK cells). The presently disclosed subject matter further provides uses of the cells and compositions comprising the same for treatment, for example, for treating neoplasms. The CD19-targeted CARs of the present disclosure have been shown to be safer and / or more potent than other CD19-targeted CARs, as evidenced, for example, by their higher persistence (see Examples 6 and 7). This greater persistence has also been shown to pose no safety risks. Thus, the CD19-targeted CAR T cells of the present disclosure can be used at lower doses than the majority of CD19-targeted CAR T cells used in current immunotherapies. The CD19-targeted CAR T cells of the present disclosure are safer and therefore are expected to have fewer side effects, including, for example, but not limited to, a lower incidence of cytokine release syndrome (CRS) and neurotoxicity (NT).

[0080] Non-limiting embodiments of the present disclosure are described herein and in the Examples.

[0081] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definition; 5.2. CD19; 5.3. Chimeric antigen receptors (CARs); 5.4. Cell; 5.5. Nucleic Acid Molecules and Vectors; 5.6. Formulation and Administration; and 5.7. Treatment Method 5.1 Definition

[0082] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the subject matter of this disclosure belongs.

[0083] As used herein, the term "about" or "approximately" refers to falling within an acceptable error range for a particular value as determined by one skilled in the art, where the acceptable error range depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0084] As used herein, the term "immunoresponsive cell" refers to a cell or its precursor or progeny that functions in an immune response. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, and stem cells that can differentiate into lymphoid cells. In certain embodiments, the immunoresponsive cell is a cell of the myeloid lineage.

[0085] As used herein, the term "activating immunoresponsive cells" refers to inducing changes in signal transduction or protein expression in cells, resulting in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade occurs. In certain embodiments, when CAR binds to an antigen, an immunological synapse is formed, including the clustering of many molecules (e.g., CD4 or CD8, CD3γ / σ / ε / ζ, etc.) near the bound receptor. This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells, increasing IL-2 production for the proliferation and expression of T cell proteins, which are master regulators, to initiate T cell-mediated immune responses.

[0086] As used herein, the term "stimulating immunoresponsive cells" refers to signals that result in a robust and sustained immune response. In certain embodiments, this occurs after immunoresponsive cell (e.g., T cell) activation or is simultaneously mediated through receptors including, but not limited to, CD28, 4-1BB, OX40, CD40, and ICOS. Receiving multiple stimulatory signals may be important for the initiation of a robust and long-lasting T cell-mediated immune response. T cells are rapidly inhibited and become unresponsive to antigens. However, the effects of these costimulatory signals can vary and generally result in increased gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that robustly respond to antigens for complete and sustained eradication.

[0087] As used herein, "complementarity-determining region" or "CDR" is defined as the amino acid sequence of the complementarity-determining region of an antibody, which is the hypervariable region of an immunoglobulin heavy chain or light chain. In certain embodiments, the "CDR" of a variable domain is the amino acid residue in the variable region, as defined by Kabat, Chothia, a combination of both Kabat and Chothia, AbM, contact, and / or conformational definitions, or any CDR determination method known in the art. Antibody CDRs can be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington, DC. The location of CDRs can also be identified as structural loop structures originally described by Chothia et al. See, e.g., Chothia et al., Nature 342: 877-883, 1989. Other approaches to CDR identification include the "AbM definition," a compromise between Kabat and Chothia and guided by Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts, as described in MacCallum et al., J. Mol. Biol., 262: 732-745, 1996. Another approach, referred to herein as the "conformational definition" of CDRs, allows for the identification of CDR positions as residues that contribute enthalpic-wise to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008.Still other CDR boundary definitions do not strictly follow one of the above approaches, but nevertheless overlap at least a portion of the Kabat CDR, but can be shortened or extended based on predictions or experimental findings that certain residues, groups of residues, or even the entire CDR do not significantly affect antigen binding. In some embodiments, CDRs can refer to CDRs defined by any approach known in the art, including a combination of approaches. The methods used herein can utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs can be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformational definitions. Generally, antibodies contain three heavy chain CDRs or CDR regions and three light chain CDRs or CDR regions within the variable region. The CDRs provide the majority of contact residues for antibody binding to the antigen or epitope. In certain embodiments, CDR regions are represented using the Kabat numbering system.

[0088] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fragment of an immunoglobulin (e.g., murine or human) heavy chain (V H ) and the variable region of the light chain (V L ) are covalently linked to the variable regions of V H ::V L It refers to a fusion protein that forms a heterodimer. H ) and light chain (V L ) is directly bonded or V H N-terminus and V L C-terminus of or V H C-terminus and V LThe heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain can be linked by a peptide-encoded linker (e.g., 10, 15, 20, or 25 amino acids) connecting the N-termini of the heavy chain variable region and the light chain variable region of the light chain variable region. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. The heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain can be linked by a linker. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80 (6): 1910-1917 (2008) and WO2014 / 087010, the entire contents of which are hereby incorporated by reference. In certain embodiments, the linker is a G4S linker.

[0089] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, provided below: GGGGSGGGGSGGGGS [SEQ ID NO: 1]

[0090] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, provided below: GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 2]

[0091] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, as provided below: GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 3]

[0092] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, as provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 4]

[0093] As used herein, the terms "substantially identical" or "substantially homologous" refer to a polypeptide or nucleic acid molecule that is at least about 50% identical or homologous to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such a sequence 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 99%, or at least about 100% identical or homologous to the amino acid or nucleic acid sequence used for comparison.

[0094] Sequence identity can be measured using sequence analysis software (e.g., Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX program sequence analysis software package). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary method for determining the degree of identity uses the BLAST program, and a probability score between e-3 and e-100 can be used, indicating closely related sequences.

[0095] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, a search can be performed against public databases, for example, to identify related sequences, using the amino acid sequence of the presently disclosed subject matter as a "query sequence." Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv703). To obtain gapped alignments for comparison purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402, can be used.When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0096] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the CD19-targeting CAR of the present disclosure (e.g., the extracellular antigen-binding domain of the CAR), including the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in the CDR regions can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained functionality (i.e., the functions described in (c) through (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues in a given sequence or CDR region are altered.

[0097] As used herein, the term "effective amount" refers to an amount sufficient to produce beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. In certain embodiments, an effective amount may be an amount sufficient to alleviate, improve, stabilize, reverse, or slow the progression of a disease, or otherwise reduce the pathological consequences of a disease. An effective amount can be determined by a physician on a case-by-case basis and within the skill of a person skilled in the art. In general, several factors are taken into consideration when determining the appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition to be treated, the severity of the condition, and the form and effective concentration of the cells to be administered.

[0098] As used herein, the term "neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or infiltration into other tissues or organs. Neoplastic growth is generally uncontrolled and progressive, occurring under conditions that prevent or halt normal cell multiplication. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, organs or tissues or cell types thereof selected from the group consisting of the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). Neoplasms can be primary tumors or primary cancers.

[0099] As used herein, the term "signal sequence" or "leader sequence" refers to a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of newly synthesized proteins that directs those proteins into the secretory pathway.

[0100] The terms "comprises" and "comprising" shall have the broad meaning given to them in U.S. patent law and may mean "includes," "including," etc.

[0101] As used herein, "treatment" refers to a clinical intervention that attempts to alter the disease course of the treated individual or cell, and can be performed for prophylaxis or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and remission or improving prognosis. Treatment can prevent the progression of the disease or disorder, thereby preventing deterioration caused by the disorder in subjects affected by, diagnosed with, or suspected of having the disorder, as well as preventing the onset of the disorder or symptoms of the disorder in subjects at risk of or suspected of having the disorder.

[0102] An "individual" or "subject," as used herein, is a vertebrate, e.g., a mammal, such as a human or non-human animal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pet animals. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys. 5.2.CD19

[0103] CD19 is a cell surface 95 kDa glycoprotein present on normal B cells early in development until they differentiate into plasma cells. CD19 is absent from other normal tissues, including pluripotent blood stem cells. CD19 is expressed on a subset of B-cell lymphomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and acute myelogenous leukemia, making it an attractive target for immunotherapy with minimal risk of autoimmune disease (other than B-cell aplasia) or irreversible bone marrow toxicity.

[0104] CD19 is a member of the immunoglobulin superfamily and a component of the cell surface signal transduction complex containing Leu13, CD81, and CD21 that positively regulates signal transduction through the B cell receptor.

[0105] In certain embodiments, the CAR of the present disclosure binds to human CD 19. In certain embodiments, the human CD19 comprises or consists of the amino acid sequence of NCBI reference number NP_001171569.1 (SEQ ID NO: 5), or a fragment thereof. SEQ ID NO: 5 is provided below: MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCLPPRDSL NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVL WHWLLRTGGW KVSAVTLAYL IFCLCSLVGI LHLQRALVLR RKRKRMTDPT RRFFKVTPPP GSGPQNQYGN VLSLPTPTSG LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVG PEEEEGEGYE EPDSEEDSEF YENDSNLGQD QLSQDGSGYE NPEDEPLGPE DEDSFSNAES YENEDEELTQ PVARTMDFLS PHGSAWDPSR EATSLAGSQS YEDMRGILYA APQLRSIRGQ PGPNHEEDAD SYENMDNPDG PDPAWGGGGR MGTWSTR [SEQ ID NO: 5]

[0106] In certain embodiments, the human CD19 comprises or consists of the amino acid sequence of NCBI reference number NP_001761.3 (SEQ ID NO: 6), or a fragment thereof. SEQ ID NO: 6 is provided below: MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCLPPRDSL NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVL WHWLLRTGGW KVSAVTLAYL IFCLCSLVGI LHLQRALVLR RKRKRMTDPT RRFFKVTPPP GSGPQNQYGN VLSLPTPTSG LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVG PEEEEGEGYE EPDSEEDSEF YENDSNLGQD QLSQDGSGYE NPEDEPLGPE DEDSFSNAES YENEDEELTQ PVARTMDFLS PHGSAWDPSR EATSLGSQSY EDMRGILYAA PQLRSIRGQP GPNHEEDADS YENMDNPDGP DPAWGGGGRM GTWSTR [SEQ ID NO: 6]

[0107] In certain embodiments, the CAR binds to the extracellular domain of CD19. In certain embodiments, the CAR binds to the extracellular domain of human CD19. In certain embodiments, the extracellular domain of human CD19 comprises or consists of amino acids 20-291 of SEQ ID NO:5. In certain embodiments, the extracellular domain of human CD19 comprises or consists of amino acids 20-291 of SEQ ID NO:6.

[0108] In certain embodiments, CD19 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 5, or a fragment thereof.

[0109] In certain embodiments, CD19 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 6, or a fragment thereof. 5.3. Chimeric Antigen Receptors (CARs)

[0110] CAR is an engineered receptor that transfers or transfers desired specificity to immune effector cells.CAR can be used to transfer the specificity of monoclonal antibody to cells, such as immune responsive cells, such as T cells or NK cells; the transfer of their coding sequences is facilitated by retroviral vectors.

[0111] There are three generations of CARs. "First generation" CARs generally consist of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain and an intracellular signaling domain fused to it. "First generation" CARs provide novel antigen recognition and, as a single fusion molecule, signal transduction via their CD3 ζ chain signaling domain, independently of HLA-mediated antigen presentation, via CD4 + T cells and CD8 + The CD19-targeted CAR can induce both activation and activation of T cells. In "second-generation" CARs, an intracellular signaling domain from a costimulatory molecule (including, but not limited to, CD28, 4-1BB, ICOS, OX40) is added to the cytoplasmic tail of the CAR to provide an additional signal to the cell. "Second-generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the CD19-targeted CAR is a first-generation CAR. In certain embodiments, the CD19-targeted CAR does not include the intracellular signaling domain of a costimulatory molecule. In certain embodiments, the CD19-targeted CAR is a second-generation CAR. In certain embodiments, the CD19-targeted CAR includes the intracellular signaling domain of a costimulatory molecule.

[0112] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds to CD19, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain is fused to the transmembrane domain, and the transmembrane domain is fused to the intracellular signaling domain. 5.3.1. Extracellular Antigen-Binding Domain of the CAR

[0113] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or is an scFv. The scFv is a human scFv, a humanized scFv, or a mouse scFv. In certain embodiments, the scFv is a human scFv. The scFv can be derived from the fusion of the heavy chain variable region and the light chain variable region of an antibody. Alternatively or additionally, the scFv can be derived from an Fab (not derived from an antibody, for example, obtained from a Fab library).

[0114] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or is Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or is F(ab)2.

[0115] Any of the foregoing molecules can be included in a fusion protein along with a heterologous sequence to form the extracellular antigen-binding domain of the CAR.

[0116] In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD19 (e.g., human CD19) at least about 1 x 10 -6 M, at least about 1 × 10 -7 M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, or at least about 1 × 10 -10 Dissociation constant of M (K d In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD19 (e.g., human CD19) at a concentration of at least about 2 x 10 -8 Dissociation constant of M (K d In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD19 (e.g., human CD19) at about 2×10 -8 M to approx. 8 x 10 -9 The dissociation constant (K d ) to join them.

[0117] In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD19 (e.g., human CD19) with a dissociation constant (K) of between about 1 nM and 50 nM, between about 5 nM and 30 nM, between about 5 nM and 25 nM, or between about 8 nM and 20 nM. d In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD19 (e.g., human CD19) with a dissociation constant (K) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, or at least about 5 nM. d ) to join them.

[0118] The binding of the extracellular antigen-binding domain of CAR can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a specific protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radiolabeled and used for radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioisotopes can be detected by means such as using a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the CD127-targeting extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the CD19-targeting human scFv is labeled with GFP.

[0119] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) is included.

[0120] In certain embodiments, V Hcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof. H comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. SEQ ID NOs: 7 to 9 are presented below. SYGMH [SEQ ID NO: 7] LIWYDGSNKYYADSVKG [SEQ ID NO: 8] PVEGLLRGFDY [SEQ ID NO: 9]

[0121] In certain embodiments, V H comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 10. For example, V H comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10. SEQ ID NO: 10 is provided below. QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSS [SEQ ID NO: 10]

[0122] In certain embodiments, V Lcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof. L comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NOs: 11 to 13 are presented below. RASQSVSSSYLA [SEQ ID NO: 11] GASSRAT [SEQ ID NO: 12] QQAGAVPIT [SEQ ID NO: 13]

[0123] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 14. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 14. SEQ ID NO: 14 is provided below. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGAVPITFGGGTKVEIK [SEQ ID NO: 14]

[0124] In certain embodiments, V Hcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13.

[0125] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:14.

[0126] V H and V L The variable regions may be linked one to the other, for example, by a linker. L -V H or V L -V H In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. Hand a V comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO: 14. L In certain embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. SEQ ID NO: 15 is provided below. In certain embodiments, the anti-CD19 scFv is referred to as "#2 scFv."

[0127] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGAVPITFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSS [SEQ ID NO: 15]

[0128] In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. SEQ ID NOs: 16 and 17 are provided below. RASQSVRSSYLA [SEQ ID NO: 16] QQLFDSPYT [SEQ ID NO: 17]

[0129] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 18. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 18. SEQ ID NO: 18 is provided below. EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQLFDSPYTFGGGTKVEIK [SEQ ID NO: 18]

[0130] In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0131] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:18.

[0132] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H and a V comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17. L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO: 18. L In certain embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19. SEQ ID NO: 19 is provided below. In certain embodiments, the anti-CD19 scFv is referred to as "#8 scFv."

[0133] IVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQLFDSPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSS [SEQ ID NO: 19]

[0134] In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NOs: 20 and 21 are provided below. GASRRAT [SEQ ID NO: 20] QQAGIPPYT [SEQ ID NO: 21]

[0135] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 22. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 22. SEQ ID NO: 22 is provided below. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGIPPYTFGGGTKVEIK [SEQ ID NO: 22]

[0136] In certain embodiments, V Hcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0137] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:22.

[0138] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 21; L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10.H and V comprising the amino acid sequence set forth in SEQ ID NO: 22. L In certain embodiments, the scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. SEQ ID NO: 23 is provided below. In certain embodiments, the anti-CD19 scFv is referred to as "#15 scFv."

[0139] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGIPPYTFGGGTKVEIK [SEQ ID NO: 23]

[0140] In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0141] In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57. SEQ ID NO: 57 is provided below. QQVDSLHPFT [SEQ ID NO: 57]

[0142] In certain embodiments, V Lcomprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 58. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 58. SEQ ID NO: 58 is provided below. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQVDSLHPFTFGGGTKVEIK [SEQ ID NO: 58]

[0143] In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57.

[0144] In certain embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:58.

[0145] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 57; L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO: 58 L In certain embodiments, the anti-CD19 scFv is referred to as "#4 scFv."

[0146] In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0147] In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59. SEQ ID NO: 59 is provided below. QQAGGVPPLT [SEQ ID NO: 59]

[0148] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 60. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 60. SEQ ID NO: 60 is provided below. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGGVPPLTFGGGTKVEIK [SEQ ID NO: 60]

[0149] In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V Lcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59.

[0150] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:60.

[0151] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 59; L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO: 60. L In certain embodiments, the anti-CD19 scFv is referred to as "#5 scFv."

[0152] In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0153] In certain embodiments, V Lcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61. SEQ ID NO: 61 is provided below. QQAGVPPLT [SEQ ID NO: 61]

[0154] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 62. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 62. SEQ ID NO: 62 is provided below. EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGVPPLTFGGGTKVEIK [SEQ ID NO: 62]

[0155] In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V Lcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0156] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:62.

[0157] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 61; L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO: 62. L In certain embodiments, the anti-CD19 scFv is referred to as "#6 scFv."

[0158] In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0159] In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63. SEQ ID NO: 63 is provided below. QQAGGVPPFT [SEQ ID NO: 63]

[0160] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 64. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 64. SEQ ID NO: 64 is provided below. EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGGVPPFTFGGGTKVEIK [SEQ ID NO: 64]

[0161] In certain embodiments, V Hcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63.

[0162] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:64.

[0163] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 63; L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10.H and V comprising the amino acid sequence set forth in SEQ ID NO: 64. L In certain embodiments, the anti-CD19 scFv is referred to as "#7 scFv."

[0164] In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0165] In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof. L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66. SEQ ID NOs: 66 and 67 are provided below. GASNRAT [SEQ ID NO: 65] QQAGVFPFT [SEQ ID NO: 66]

[0166] In certain embodiments, V L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence set forth in SEQ ID NO: 67. For example, V L comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 67. In certain embodiments, V L comprises the amino acid sequence set forth in SEQ ID NO: 67. SEQ ID NO: 67 is provided below. EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGVFPFTFGGGTKVEIK [SEQ ID NO: 67]

[0167] In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66 or a conservative modification thereof. H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0168] In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 10, and V L comprises the amino acid sequence set forth in SEQ ID NO:67.

[0169] In certain embodiments, V L is located at the N-terminus of the extracellular antigen-binding domain, i.e., V H and V L is, from the N-terminus to the C-terminus, V L -V HIn certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 65, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 66; L In certain embodiments, the scFv is a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO: 67 L In certain embodiments, the anti-CD19 scFv is referred to as "#1 scFv."

[0170] In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0171] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 is set forth in SEQ ID NO: 68. SEQ ID NO: 68 is provided below.

[0172] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTGATATGGTATGATGGAAGTAATAAATACTATGCAG ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGCCAGTGGAAGGACTATTAAGAGGATTCGATTACTGGGGACAGGGTACATTGGTCACCGTCTCCTCA [SEQ ID NO: 68]

[0173] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 14 is set forth in SEQ ID NO: 69. SEQ ID NO: 69 is provided below.

[0174] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGAGCCGTCCCTATCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 69]

[0175] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 70. SEQ ID NO: 70 is provided below.

[0176] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGGAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCTCTTCGACAGTCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 70]

[0177] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22 is set forth in SEQ ID NO: 71. SEQ ID NO: 71 is provided below.

[0178] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGAAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGGCCGGCATCCCCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 71]

[0179] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 58 is set forth in SEQ ID NO: 72. SEQ ID NO: 72 is provided below.

[0180] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGC CACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGTCGACAGTCTCCATCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 72]

[0181] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 60 is set forth in SEQ ID NO: 73. SEQ ID NO: 73 is provided below.

[0182] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGC CACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGAGGCGTCCCTCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 73]

[0183] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 62 is set forth in SEQ ID NO: 74. SEQ ID NO: 74 is provided below.

[0184] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGGAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGAGTCCCCCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 74]

[0185] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 64 is set forth in SEQ ID NO: 75. SEQ ID NO: 75 is provided below.

[0186] GAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGC CACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGAGGCGTCCCTCCTTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 75]

[0187] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 67 is set forth in SEQ ID NO: 76. SEQ ID NO: 76 is provided below.

[0188] GAAATTGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAACAGGG CCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGAGTCTTCCCTTTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA [SEQ ID NO: 76]

[0189] In certain embodiments, V H and V Lare linked via a linker. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0190] A V that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to a particular sequence (e.g., SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the designated sequence(s), but retain the ability to bind to a target antigen (e.g., mesothelin). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within a particular sequence (e.g., SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO: 67). In certain embodiments, the substitutions, insertions, or deletions are in regions outside the CDRs of the extracellular antigen-binding domain (e.g., within the FRs). In certain embodiments, the extracellular antigen-binding domain is selected from SEQ ID NOs: 10, 14, 18, 22, 59, 61, 63, 65, and 68. H and / or V L and including post-translational modifications of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, or SEQ ID NO:67.

[0191] Furthermore, the extracellular antigen-binding domain may contain a leader or signal peptide that directs the nascent protein into the endoplasmic reticulum. The signal peptide or leader may be essential when the CAR is glycosylated and anchored to the cell membrane. The signal sequence or leader may be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids in length) present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway. In certain embodiments, the signal peptide is covalently attached to the 5' end (N-terminus) of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a CD8 polypeptide, for example, the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the signal peptide is generated from the antibody from which it is derived. In certain embodiments, the signal peptide for #2 scFv is set forth in SEQ ID NO: 54. In certain embodiments, the signal peptide for #8 scFv is set forth in SEQ ID NO: 54. In certain embodiments, the signal peptide for #15 scFv is set forth in SEQ ID NO: 55. MDMRVPAQLLGLLLLWLPDTRC [SEQ ID NO: 54] MEFGLSWVFLVALLRGVQC [SEQ ID NO: 55] 5.3.2. CAR Transmembrane Domain

[0192] In certain embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors cluster and transmit a signal to the cell. In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of CD8, a native or modified transmembrane domain of CD28, a native or modified transmembrane domain of CD3ζ, a native or modified transmembrane domain of CD4, a native or modified transmembrane domain of 4-1BB, a native or modified transmembrane domain of OX40, a native or modified transmembrane domain of ICOS, a native or modified transmembrane domain of CD84, a native or modified transmembrane domain of CD166, a native or modified transmembrane domain of CD8a, a native or modified transmembrane domain of CD8b, a native or modified transmembrane domain of ICAM-1, a native or modified transmembrane domain of CTLA-4, a native or modified transmembrane domain of CD27, a native or modified transmembrane domain of CD40, a native or modified transmembrane domain of NKGD2, or a combination thereof.

[0193] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a portion thereof. The CD28 polypeptide may comprise or consist of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical or homologous to the sequence having NCBI reference number NP_006130 (SEQ ID NO: 24) or a fragment thereof, and / or may contain up to one, or up to two, or up to three conservative amino acid substitutions, as appropriate. In certain embodiments, a CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:24 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, and / or up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, or up to about 220 amino acids in length. In certain embodiments, a CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 154-179, 150-200, 153-179, or 200-220 of SEQ ID NO:24. In certain embodiments, the transmembrane domain of a CAR comprises a CD28 polypeptide that comprises or consists of the amino acid sequence of amino acids 154-179 of SEQ ID NO:24. SEQ ID NO:24 is provided below. MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS [SEQ ID NO: 24]

[0194] Amino acids 154-179 of an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:24 are set forth in SEQ ID NO:25, which is provided below. TGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG [SEQ ID NO: 25]

[0195] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD28 or a portion thereof. The CD28 polypeptide may consist of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical or homologous to the sequence having NCBI reference number NP_031668.3 (SEQ ID NO: 26) or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 26 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-200, 151-177, or 200-218 of SEQ ID NO: 26. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 151-177 of SEQ ID NO: 26. SEQ ID NO: 26 is provided below: MTLRLLFLAL NFFSVQVTEN KILVKQSPLL VVDSNEVSLS CRYSYNLLAK EFRASLYKGV NSDVEVCVGN GNFTYQPQFR SNAEFNCDGD FDNETVTFRL WNLHVNHTDI YFCKIEFMYP PPYLDNERSN GTIIHIKEKH LCHTQSSPKL FWALVVVAGV LFCYGLLVTV ALCVIWTNSR RNRLLQSDYM NMTPRRPGLT RKPYQPYAPA RDFAAYRP [SEQ ID NO: 26]

[0196] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the sequence having NCBI reference number NP_001139345.1 (SEQ ID NO: 27) or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 27 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 235 amino acids in length. Alternatively or additionally, in certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO: 27. In certain embodiments, the transmembrane domain of a CAR comprises a CD8 polypeptide comprising or consisting of the amino acid sequence of amino acids 137-209 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below.

[0197] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV [SEQ ID NO: 27]

[0198] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the sequence having NCBI reference number AAA92533.1 (SEQ ID NO: 28) or a fragment thereof, and / or optionally contains up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 28 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. Alternatively or additionally, in certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 28. In certain embodiments, the transmembrane domain of a CAR comprises a CD8 polypeptide comprising or consisting of the amino acid sequence of amino acids 151-219 of SEQ ID NO: 28. SEQ ID NO: 28 is provided below. MASPLTRFLS LNLLLMGESI ILGSGEAKPQ APELRIFPKK MDAELGQKVD LVCEVLGSVS QGCSWLFQNS SSKLPQPTFV VYMASSHNKI TWDEKLNSSK LFSAVRDTNN KYVLTLNKFS KENEGYYFCS VISNSVMYFS SVVPVLQKVN STTTKPVLRT PSPVHPTGTS QPQRPEDCRP RGSVKGTGLD FACDIYIWAP LAGICVAPLL SLIITLICYH RSRKRVCKCP RPLVRQEGKP RPSEKIV [SEQ ID NO: 28]

[0199] In certain non-limiting embodiments, the CAR further comprises a spacer region linking the extracellular antigen-binding domain and the transmembrane domain. The spacer region can be sufficiently flexible to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition while preserving the activity of the CAR.

[0200] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or a combination thereof. The hinge / spacer region may be a hinge region derived from an IgG1, or an immunoglobulin CH2CH3 region and a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO:24 or SEQ ID NO:26), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO:27 or SEQ ID NO:28), a variation of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identical or homologous thereto, or a synthetic spacer sequence.

[0201] In certain embodiments, the hinge domain of the CAR comprises the native or modified hinge region of CD28. In certain embodiments, the hinge domain of the CAR comprises the native hinge region of CD28. In certain embodiments, the hinge domain of the CAR comprises the amino acid sequence of amino acids 114-153 of SEQ ID NO: 24. Amino acids 114-153 of an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24 is set forth in SEQ ID NO: 56, which is provided below. ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTT [SEQ ID NO: 56] 5.3.3. CAR Intracellular Signaling Domain

[0202] In certain embodiments, the CAR comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("natural") CD3ζ contains three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2, ​​and BRS3). CD3ζ transmits activation signals to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the primary transmitter of signals from endogenous TCRs.

[0203] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence having NCBI reference number NP_932170 (SEQ ID NO:29) or a fragment thereof, and / or optionally contains up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:29 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, native CD3ζ comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 29. In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ comprising or consisting of the amino acid sequence of amino acids 52-164 of SEQ ID NO: 29. SEQ ID NO: 29 is provided below: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR [SEQ ID NO: 29]

[0204] In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 30. SEQ ID NO: 30 is provided below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 30]

[0205] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises one, two, or three ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1. In certain embodiments, the native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31. QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 31]

[0206] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:31 is set forth in SEQ ID NO:32, which is provided below. CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA [SEQ ID NO: 32]

[0207] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM1. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33, as provided below. QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 33]

[0208] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:33 is set forth in SEQ ID NO:34, which is provided below. CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA [SEQ ID NO: 34]

[0209] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, as provided below. QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 35]

[0210] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:35 is set forth in SEQ ID NO:36, which is provided below. CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA [SEQ ID NO: 36]

[0211] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant. In certain embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, as provided below. QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 37]

[0212] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:37 is set forth in SEQ ID NO:38, which is provided below. CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAA [SEQ ID NO: 38]

[0213] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39, as provided below. HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 39]

[0214] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:39 is set forth in SEQ ID NO:40, which is provided below. CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG [SEQ ID NO: 40]

[0215] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41, as provided below. HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 41]

[0216] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:41 is set forth in SEQ ID NO:42, which is provided below. CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAG[SEQ ID NO: 42]

[0217] Various modified CD3ζ polypeptides and CARs comprising modified CD3ζ polypeptides are disclosed in International Patent Application Publication No. WO2019 / 133969, which is hereby incorporated by reference in its entirety.

[0218] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:31, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:37, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:41. In certain embodiments, the CAR is referred to as "1XX." In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:43. SEQ ID NO:43 is provided below: RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLFN ELQKDKMAEA FSEIGMKGER RRGKGHDGLF QGLSTATKDT FDALHMQALP PR [SEQ ID NO: 43]

[0219] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises or consists of an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to SEQ ID NO: 43 or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions.

[0220] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:43 is set forth in SEQ ID NO:44, which is provided below.

[0221] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC TGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 44]

[0222] Another exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:43 is set forth in SEQ ID NO:45, which is provided below.

[0223] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC TGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 45]

[0224] In certain embodiments, the intracellular signaling domain of CAR further comprises at least one costimulatory signaling region.In certain embodiments, at least one costimulatory region comprises a costimulatory molecule or a portion thereof.In certain embodiments, at least one costimulatory region comprises the intracellular domain of at least one costimulatory molecule or a portion thereof.

[0225] As used herein, "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand, which can result in an efficient response of lymphocytes to antigens. In certain embodiments, costimulatory molecules can result in optimal lymphocyte activation. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, and combinations thereof. A costimulatory molecule can bind to a costimulatory ligand. A costimulatory ligand is a protein that is expressed on the cell surface and, upon binding to its receptor, generates a costimulatory response, i.e., an intracellular response that enables the stimulation that occurs when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen. As an example, a 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB, thereby combining with a CAR signal to activate the CAR. +It provides an intracellular signal that induces the effector cell function of the T cell. In certain embodiments, at least one costimulatory signaling region comprises the intracellular signaling domain of CD28 or a portion thereof, the intracellular domain of 4-1BB or a portion thereof, the intracellular domain of OX40 or a portion thereof, the intracellular domain of ICOS or a portion thereof, or the intracellular domain of DAP-10 or a portion thereof.

[0226] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of a CD28 polypeptide, e.g., CD28, or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human CD28, or a portion thereof.

[0227] In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 24, or a fragment thereof, and / or optionally comprises up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 24 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region comprises or consists of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 24. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 180-220 of SEQ ID NO: 24.

[0228] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180-220 of SEQ ID NO:24 is set forth in SEQ ID NO:46, which is provided below. AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 46]

[0229] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 26, or a fragment thereof, and / or optionally contains up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 26 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, a CD28 polypeptide comprised in a costimulatory signaling region comprises or consists of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 26. In certain embodiments, a costimulatory signaling region of a CAR of the disclosure comprises a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 178-218 of SEQ ID NO: 26.

[0230] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., the intracellular domain of 4-1BB, or a portion thereof. In certain embodiments, the costimulatory signaling region comprises the intracellular domain of human 4-1BB, or a portion thereof. In certain embodiments, the 4-1BB comprised in the costimulatory signaling region comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical or homologous to the sequence having NCBI reference number NP_001552 (SEQ ID NO:47) or a fragment thereof, and / or optionally comprises up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB included in the costimulatory signaling region comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:47 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 200, or up to about 255 amino acids in length. In certain embodiments, the 4-1BB polypeptide included in the costimulatory signaling region comprises or consists of the amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO:47. In certain embodiments, the costimulatory signaling region comprises a 4-1BB polypeptide that comprises or consists of the amino acid sequence of amino acids 214-255 of SEQ ID NO:46. SEQ ID NO:47 is provided below.

[0231] MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL [SEQ ID NO: 47]

[0232] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that includes the intracellular domains or portions thereof of two or more costimulatory molecules, e.g., the intracellular domain or portion thereof of CD28 and the intracellular domain or portion thereof of 4-1BB, or the intracellular domain or portion thereof of CD28 and the intracellular domain or portion thereof of OX40. 5.3.4. CAR Instances

[0233] In certain embodiments, the CAR comprises (a)(i) a V CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H and (ii) a V comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 13. L(b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., a hinge domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 114-153 of SEQ ID NO: 24); (c) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 154-179 of SEQ ID NO: 24), and (d) an intracellular signaling domain comprising (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, e.g., one consisting of the amino acid sequence set forth in SEQ ID NO: 43), and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 180-220 of SEQ ID NO: 24). In certain embodiments, V H and V L are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the CAR is designated as "CAR#2". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 48, which is provided below.

[0234] MDMRVPAQLLGLLLLWLPDTRCEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGAVPITFGGG TKVEIKGGGGSGGGGSGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGL LRGFDYWGQGTLVTVSSRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 48]

[0235] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:48 is set forth in SEQ ID NO:49, which is provided below.

[0236]

[0237] In certain embodiments, the CAR comprises (a)(i) a V CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H and (ii) a V comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 17. L (b) an extracellular antigen-binding domain comprising: (a) a hinge domain comprising a CD28 polypeptide (e.g., a hinge domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 114-153 of SEQ ID NO: 24); (c) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 114-179 of SEQ ID NO: 24); and (c) an intracellular signaling domain comprising: (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, e.g., one consisting of the amino acid sequence set forth in SEQ ID NO: 43); and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 180-220 of SEQ ID NO: 24). In certain embodiments, V H and V L are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the CAR is designated as "CAR#8". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 50, which is provided below.

[0238] MDMRVPAQLLGLLLLWLPDTRCEIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQ QKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQLFDSPYT FGGGTKVEIKGGGGSGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CAKPVEGLLRGFDYWGQGTLVTVSSRAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDAL HMQALPPR [SEQ ID NO: 50]

[0239] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:50 is set forth in SEQ ID NO:51, which is provided below.

[0240]

[0241] In certain embodiments, the CAR comprises (a)(i) a V CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H and (ii) a V comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 21. L (b) an extracellular antigen-binding domain comprising: (a) a hinge domain comprising a CD28 polypeptide (e.g., a hinge domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 114-153 of SEQ ID NO: 24); (c) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 114-179 of SEQ ID NO: 24); and (c) an intracellular signaling domain comprising: (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, e.g., one consisting of the amino acid sequence set forth in SEQ ID NO: 43); and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof, e.g., a CD28 polypeptide consisting of amino acids 180-220 of SEQ ID NO: 24). In certain embodiments, V H and V L are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, V H and V L is, from the N-terminus to the C-terminus, V L -V H In certain embodiments, the CAR is designated as "CAR#15". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 51, which is provided below.

[0242] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAP GKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVE GLLRGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRAS QSVSSSYLAWYQQKPGQAPRLLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQAGIPPYTFGGGTKVEIKRAAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP GPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQ ALPPR [SEQ ID NO: 52]

[0243] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:52 is set forth in SEQ ID NO:53, which is provided below.

[0244] 5.4.Cells

[0245] The subject matter of the present disclosure provides a cell comprising the CD19-targeting CAR of the present disclosure (for example, as disclosed in Section 5.3).In certain embodiments, the cell is selected from the group consisting of lymphoid lineage cells and myeloid lineage cells.In certain embodiments, the cell is an immunoresponsive cell.In certain embodiments, the immunoresponsive cell is a lymphoid lineage cell.

[0246] In certain embodiments, the cells are lymphoid lineage cells. Lymphoid lineage cells can produce antibodies, regulate the cellular immune system, detect foreign substances in the blood, detect cells that are foreign to the host, etc. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells that can differentiate into lymphoid cells. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0247] In certain embodiments, the cells are T cells. T cells are lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the presently disclosed subject matter may be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMR cells), regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introducing a CAR. In certain embodiments, the immunoresponsive cells are T cells. T cells are CD4 + T cells or CD8 +In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + The cell is a T cell. In certain embodiments, the cell is a T cell, and the CD19 targeting CAR of the present disclosure is integrated into the locus in the genome of the T cell. Non-limiting examples of the locus include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. The method of targeting CAR to the site in the genome of T cell is disclosed in WO2017180989 and Eyquem et al., Nature. (2017 Mar 2); 543 (7643): 113-117, both of which are incorporated by reference in their entirety.

[0248] In certain embodiments, the cells are NK cells. Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not need to be pre-activated to exert a cytotoxic effect on their target cells. In certain embodiments, the cells are genetically modified NK cells. In certain embodiments, the cells are edited NK cells. In certain embodiments, the cells are NK cells derived from stem cells. In certain embodiments, the cells are NK cells derived from pluripotent stem cells. In certain embodiments, the cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0249] The cells (e.g., T cells or NK cells) may be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered precursor or stem cells.

[0250] The subject cells of the present disclosure can be myeloid lineage cells. Non-limiting examples of myeloid lineage cells include stem cells that can differentiate into monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and myeloid cells. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0251] In certain embodiments, cells can be transduced with a CD19-targeted CAR of the present disclosure, thus allowing the cells to express the CD19-targeted CAR. 5.5. Nucleic Acid Molecules and Vectors

[0252] The presently disclosed subject matter provides nucleic acid molecules encoding the CD19-targeting CARs of the present disclosure (e.g., those disclosed in Section 5.3). Cells containing such nucleic acid molecules are also provided.

[0253] In certain embodiments, the nucleic acid molecule further comprises a promoter operably linked to the CD19-targeting CAR of the present disclosure.

[0254] In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the group consisting of an elongation factor (EF)-1 promoter, a cytomegalovirus immediate early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter, and a ubiquitin C promoter. In certain embodiments, the endogenous promoter is selected from a TCR alpha promoter, a TCR beta promoter, and a beta2-microglobulin promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a 4-1BB promoter, a PD1 promoter, and a LAG3 promoter.

[0255] The presently disclosed subject matter also provides vectors comprising the nucleic acid molecules of the present disclosure.

[0256] Nucleic acid molecules can be delivered to cells by methods known in the art or as described herein.Cell genetic modification can be achieved by transducing recombinant DNA constructs into substantially homogeneous cell compositions.In certain embodiments, retroviral vectors (for example, gammaretroviral vectors or lentiviral vectors) are used to introduce DNA constructs into cells.For example, the polynucleotide encoding CAR can be cloned into retroviral vectors, and expression can be driven from its endogenous promoter, from retroviral long terminal repeats, or from a promoter specific to the target cell type of interest.Non-viral vectors can also be used.

[0257] For the initial genetic modification of cells to include the CD19-targeting CAR of the present disclosure, a retroviral vector can be used for transduction, but any other suitable viral vector or non-viral delivery system can be used. CARs can be constructed as a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, such as FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A, and F2A peptides). If the capsid protein is functional for infecting human cells, the combination of a retroviral vector with an appropriate packaging system is also suitable.Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., (1985) Mol Cell Biol (1985); 5: 431-437); PA317 (Miller, et al., Mol Cell Biol (1986); 6: 2895-2902); and CRIP (Danos et al., Proc Natl Acad Sci USA (1988); 85: 6460-6464).For example, particles pseudotyped against VSVG, RD114, or GALV envelopes and any other non-amphotropic particles known in the art are also suitable.

[0258] Possible methods of transduction include direct co-culturing of cells with producer cells (Bregni et al., Blood (1992); 80: 1418-1422), or culturing with viral supernatant alone or concentrated vector stocks, with or without appropriate growth factors and polycations (Xu et al., Exp Hemat (1994); 22: 223-230; and Hughes et al. J Clin Invest (1992); 89: 1817).

[0259] Other transduction virus vectors can also be used to modify cells.In certain embodiments, the vector selected is one that shows high-efficiency infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8: 423-430, 1997; Kido et al., Current Eye Research 15: 833-844, 1996; Bloomer et al., Journal of Virology 71: 6641-6649, 1997; Naldini et al., Science 272: 263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94: 10319, 1997).Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see, e.g., Miller, Human Gene Thera (1990); 15-14; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques (1988); 6: 608-614; Tolstoshev et al., Cur Opin Biotechnol (1990); 1: 55-61; Sharp, The Lancet (1991); 337: 1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36: 311-22, 1987; Anderson, Science (1984); 226: (See also Moen, Blood Cells 17: 401-409; Miller et al., Biotechnol (1989); 7: 980-90; LeGal La Salle et al., Science (1993); 259: 988-90; and Johnson, Chest (1995) 107: 77S-83S). Retroviral vectors have been particularly well developed and used in clinical settings (Rosenberg et al., N Engl J Med (1990); 323: 370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0260] Non-viral methods can also be used for genetic modification of cells.For example, nucleic acid molecules can be introduced into cells by administering nucleic acid in the presence of lipofection (Feigner et al., Proc Natl Acad Sci USA (1987);84:7413;Ono et al., Neurosci Lett (1990);17:259;Brigham et al., Am J Med Sci (1989);298:278;Staubinger et al., Methods in Enzymol (1983);101:512;Wu et al., J Biol Chem (1988);263:14621;Wu et al., J Biol Chem (1989);264:16985) or by microinjection under surgical conditions (Wolff et al., Science (1990);247:1465). Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes are also potentially useful for delivering DNA to cells. Normal nucleic acids can also be transferred ex vivo into culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then the cells (or their progeny) can be injected into target tissues or injected systemically, thereby achieving the transplantation of normal genes into the target affected tissues. Transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALENs, CRISPRs) can also be used to extract or obtain recombinant receptors. Transient expression can be obtained by RNA electroporation.

[0261] Any targeted genome editing method can also be used to deliver the CD19 targeting CAR of the present disclosure into cells.In certain embodiments, the CD19 targeting CAR of the present disclosure is delivered using CRISPR system.In certain embodiments, the CD19 targeting CAR of the present disclosure is delivered using zinc finger nuclease.In certain embodiments, the CD19 targeting CAR of the present disclosure is delivered using TALEN system.

[0262] The clustered regularly interspaced short interdigitated repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains RNA that guides Cas9 to a precise section of host DNA along with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process, allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid for transfection into target cells. The crRNA is the sequence that Cas9 uses to identify and directly bind to target DNA within the cell, and therefore needs to be designed for each application. The repair template containing the CAR expression cassette must also be designed for each application, overlapping the sequences on both sides of the cut and encoding the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be conjugated with the Cas9 gene into a plasmid for transfection into cells.

[0263] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is by combining small zinc finger "modules" with known specificities. The most common cleavage domain for ZFNs is the nonspecific cleavage domain derived from the type II restriction endonuclease FokI. ZFNs can be used to insert a CAR expression cassette into a genome by using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying the CAR expression cassette. Once the targeted sequence is cleaved by the ZFN, the HR machinery searches for a DNA template homologous to the damaged chromosome and then copies the sequence of the template between the two cut ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0264] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific sequences in DNA. TALEN systems operate on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain. Transcription activator-like effectors (TALEs) consist of a repeating motif of 33–34 amino acids, with two variable portions that have strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind to a desired DNA sequence, thereby guiding the nuclease to cut at a specific location within the genome. cDNA expression for use in polynucleotide therapy can be directed by any appropriate promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), metallothionein promoter, or ubiquitin C promoter) and regulated by any appropriate mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, the expression of nucleic acid can be directed using enhancers that are known to preferentially direct gene expression in specific cell types.The enhancers used can include, but are not limited to, those characterized as tissue-specific or cell-specific enhancers.Alternatively, when genomic clones are used as therapeutic constructs, regulation can be mediated by homologous regulatory sequences, or if desired, by regulatory sequences from heterologous sources, including any of the promoters or regulatory elements described above.

[0265] The method for delivering genome editing agent / system can vary as needed.In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids.In certain embodiments, the components are delivered by viral vector.Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impale infection, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, pseudotyped viral vector envelope protein, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (for example, oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides). 5.6. Formulation and Administration

[0266] The presently disclosed subject matter also provides a composition comprising the cells of the present disclosure, including the CD19-targeting CAR of the present disclosure. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0267] The compositions containing the cells of the present disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, and can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can contain a carrier, which can be, for example, a solvent or dispersion medium containing water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0268] A composition comprising the cells of the present disclosure can be provided to a subject systemically or directly to induce and / or enhance an immune response to an antigen and / or treat and / or prevent a neoplasm. In certain embodiments, the cells of the present disclosure or a composition comprising the same are directly injected into a target organ (e.g., an organ affected by a neoplasm). Alternatively, the cells of the present disclosure or a composition comprising the same are provided to a target organ indirectly, for example, by administration into the circulatory system (e.g., tumor vasculature). To increase the production of cells in vitro or in vivo, an expansion and differentiation inducer can be provided before, during, or after administration of the cells or composition.

[0269] The number of cells administered can vary depending on the subject being treated. In certain embodiments, about 10 4 From about 10 10 Between pieces, approximately 10 4 From about 10 7 Between pieces, approximately 10 5 From about 10 7 Between pieces, approximately 10 5 From about 10 9 Between pieces, or about 10 6 From about 10 8 Between about 1 x 10 cells of the present disclosure are administered to a subject. In certain embodiments, between about 1 x 10 cells of the present disclosure are administered to a subject. 6 From about 5 x 10 8 Between about 1 x 10 cells of the present disclosure are administered to a subject. Even smaller numbers of cells can be administered for greater efficacy. Typically, at least about 1 x 10 cells are administered. 5 The final dose was approximately 1 × 10 cells. 10 In certain embodiments, at least about 1 x 10 5 pieces, 5×10 5 pieces, 1×10 6 pieces, about 5×10 6 pieces, about 1×10 7 pieces, approximately 2.5×10 7 pieces, about 5×10 7 pieces, about 1×10 8 pieces, approximately 1.5×10 8 pieces, approximately 2×10 8pieces, or approximately 5 x 10 8 In certain embodiments, about 1 x 10 cells of the present disclosure are administered to a subject. 6 The cells of the present disclosure are administered to the subject.The precise determination of what is considered as effective dose can depend on the factors specific to each subject, including subject size, age, sex, weight and specific subject condition.Dosage can be easily determined by those skilled in the art from the present disclosure and knowledge in the art.

[0270] The cells and compositions of the present disclosure can be administered to a subject by any method known in the art, including, but not limited to, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration. The cells of the present disclosure can be administered in any physiologically acceptable vehicle, usually intravascularly, but can also be introduced into bone or other convenient sites (e.g., the thymus) where the cells can find a suitable site for regeneration and differentiation. 5.7. Treatment Method

[0271] The presently disclosed subject matter provides various methods using the cells of the present disclosure or compositions comprising them. The cells of the present disclosure and compositions comprising them can be used for therapy or medicine. For example, the presently disclosed subject matter provides a method for inducing and / or enhancing an immune response in a subject in need thereof. The cells of the present disclosure and compositions comprising them can be used to reduce tumor burden in a subject. The cells of the present disclosure and compositions comprising them can reduce the number of tumor cells, shrink tumor size, and / or eradicate tumors in a subject. The cells of the present disclosure and compositions comprising them can be used to treat and / or prevent neoplasms in a subject. The cells of the present disclosure and compositions comprising them can be used to prolong the survival of a subject suffering from a neoplasm. In certain embodiments, each of the above methods involves administering the cells of the present disclosure or a composition comprising them (e.g., a pharmaceutical composition) to achieve a desired effect, such as alleviating an existing condition or preventing recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided by bolus or continuous perfusion.

[0272] In certain embodiments, the tumor and / or neoplasm is associated with CD19. In certain embodiments, the tumor and / or neoplasm expresses CD19. In certain embodiments, the tumor and / or neoplasm overexpresses CD19. In certain embodiments, the tumor and / or neoplasm that can be treated with the cells and compositions of the present disclosure is a blood cancer. Non-limiting examples of blood cancers include multiple myeloma, leukemia, and lymphoma. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. The lymphoma may be Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the lymphoma is a B-cell lymphoma (BCL).

[0273] In certain embodiments, the tumor and / or neoplasm is a B-cell malignancy. Non-limiting examples of B-cell malignancies include B-cell lymphoma (BCL), B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma. B-cell lymphomas include B-cell non-Hodgkin's lymphoma (NHL) and B-cell Hodgkin's lymphoma. In certain embodiments, the tumor and / or neoplasm is a B-cell lymphoma. In certain embodiments, the B-cell lymphoma is a relapsed or refractory (R / R) B-cell lymphoma.

[0274] In certain embodiments, the subject is a human subject.The subject may have an advanced form of the disease, and in this case, the purpose of treatment may include alleviating or reversing the progression of the disease and / or improving side effects.The subject may have a history of the condition that has already been treated, and in this case, the purpose of treatment generally includes reducing the risk of recurrence or delaying recurrence.

[0275] As a result of surface expression of the CD19-targeted CAR of the present disclosure, the adoptively transferred cells are endowed with enhanced and selective cytolytic activity at the tumor site. Furthermore, following their localization and proliferation in the tumor, they transform the tumor site into an environment highly conducive to a wide range of cells involved in the physiological anti-tumor response.

[0276] To prevent or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as tumor cells, leading to a similar outcome as GvHD, further modifications can be introduced into the cells of the present disclosure. A potential solution to this problem is to engineer a suicide gene into the cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase-9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide may enable T cells to be eliminated by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently linked upstream of the CD19-targeted CAR. The suicide gene can be included in a vector containing a nucleic acid encoding the CD19-targeted CAR of the present disclosure. Thus, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug (e.g., AP1903, which can activate iCasp-9) during malignant T cell transformation (e.g., GVHD) induces apoptosis in cells expressing CD19-targeted CARs with activated suicide genes. Incorporation of a suicide gene into the CD19-targeted CARs of the present disclosure provides an additional level of safety by allowing the elimination of the majority of receptor-expressing cells in a very short period of time. Cells of the present disclosure with incorporated suicide genes can be preemptively eliminated at a given time point after cell injection or can be eradicated upon signs of toxicity. [Example]

[0277] 7. Working Example The subject matter of the present disclosure will be better understood by reference to the following examples, which are offered by way of illustration, but not limitation, of the subject matter of the present disclosure. Example 1 Identification and characterization of anti-CD19 antibodies

[0278] In this example, the V of the scFv disclosed herein H and V L We demonstrate the characterization of anti-CD19 antibodies, including: The human anti-CD19 antibody was derived from and produced in Adimab yeast. The antigen was biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific, Cat#21425). The antigen was concentrated to approximately 1 mg / mL, and the buffer was exchanged with PBS before adding a 1:7.5 molar ratio of biotinylation reagent. The mixture was kept overnight at 4°C, followed by a further buffer exchange to remove free biotin in solution. Biotinylation was confirmed by binding of the labeled protein to a streptavidin sensor on a ForteBio.

[0279] About 10 each 9 Eight naive human synthetic yeast libraries with a diversity of 100 were expanded as previously described (see, e.g., Y. Xu et al, PEDS 26 (10), 663-70 (2013); WO2009036379; WO2010105256; and WO2012009568).

[0280] For the first two rounds of selection, magnetic bead sorting was performed using the Miltenyi MACS system as previously described (see, e.g., Siegel et al., J Immunol Methods 286 (1-2), 141-153 (2004)). Briefly, yeast cells (approximately 10 cells) were cultured in a 500-well plate. 10Yeast (1000 cells / library) were incubated with biotinylated antigen in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 30 minutes at 30°C. After one wash with 40 mL of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, and Streptavidin MicroBeads (500 μl) were added to the yeast and incubated for 15 minutes at 4°C. The yeast were then pelleted, resuspended in 5 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 5 mL, the column was washed three times with 3 mL of wash buffer. The column was then removed from the magnetic field, and the yeast were eluted with 5 mL of growth medium and then grown overnight.

[0281] The third round of selection was performed using flow cytometry (FACS). Approximately 2 x 10 7 The yeast cells were pelleted, washed three times with wash buffer, and incubated with 100–200 nM biotinylated antigen at 30°C under equilibrium conditions. Rounds 4 and 5 of selection were performed by incubating biotinylated NALM-6 and Raji cells with the selected yeast output from round 3 FACS. After incubation, prewashed M-280 Strepavidin Dynabeads (Cat# 60210) were added to the yeast / mammalian cell complexes and incubated. The complexes were then separated using a DynaMag-2 magnet, and unbound supernatant was removed. The bead / cell complexes were washed three times with 1 mL of selection buffer. The captured complexes were then transferred to flasks containing yeast growth medium for expansion. In round 6 of selection, the expanded yeast were subjected to either an additional round of NALM-6 / Raji cell selection, selection with 100 nM recombinant CD19 antigen, or negative selection with polyspecific reagent (PSR) to remove nonspecific antibodies.

[0282] For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see, e.g., Y. Xu et al., PEDS 26 (10), 663-70 (2013)). Yeast cells were then washed twice with wash buffer and stained with a 1:100 dilution of goat F(ab')2 anti-human kappa-FITC (LC-FITC) (Southern Biotech, Cat# 2062-02) and a 1:500 dilution of streptavidin-AF633 (SA-633) (Life Technologies, Cat# S21375) or a 1:50 dilution of extravidin-phycoerthyrin (EA-PE) (Sigma-Aldrich, Cat# E4011) secondary reagent for 15 minutes at 4°C. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a sorting tube with a strainer cap. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined to select antibodies with the desired characteristics. Selection rounds were repeated until a population possessing all the desired characteristics was obtained. After the final round of sorting, yeast was plated, and individual colonies were picked for characterization. Light chain diversification

[0283] Heavy chains from the naive output were used to prepare a light chain diversified library for further rounds of selection. Heavy chain plasmids were extracted from yeast, expanded in E. coli, and subsequently purified from E. coli, yielding 5×10 6These libraries were then transformed into a diverse light chain library. These libraries were subjected to a fourth round of selection, as described above: one round of MACS, two rounds of cell selection using Raji or NALM-6 cells, followed by FACS selection using recombinant CD19 antigen. Specific to light chain diversification, Raji and NALM-6 cell selection was combined with an initial negative selection using engineered Raji and NALM-6 cells that had undergone targeted gene knockout of the CD19 gene. After depletion of CD19 knockout cells, positive selection was performed using engineered Raji and NALM-6 cells that both express and overexpress endogenous CD19. In different FACS selection rounds, the libraries were evaluated for (multispecific reagent) PSR binding, species cross-reactivity, and affinity pressure by antigen titration. Selection was performed to obtain populations with the desired characteristics. Individual colonies from each of the above FACS selection rounds were picked for sequencing and characterization. Antibody production and purification

[0284] Yeast clones were grown to saturation and then induced for 48 hours with shaking at 30°C. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid, pH 2.0. ForteBio K D measurement

[0285] These anti-CD19 antibodies were assayed for binding affinity to soluble CD19 using the ForteBio Octet system (Octet RED384, generally as previously described (e.g., Estep et al., Mabs 5 (2), 270-278). (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG onto the AHC sensor online. The antibody was immobilized on anti-human IgG pins and bound to a soluble CD19-HSA fusion protein (CD19 extracellular domain fused to human serum albumin). The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM of soluble CD19-HSA fusion protein antigen for 3 minutes, and then transferred to assay buffer for 3 minutes for dissociation rate measurements. All kinetics were analyzed using a 1:1 binding model. A summary of the binding kinetics is presented in Table 1. The affinities of the exemplary antibodies ranged from 8 nM to 20 nM. All anti-CD19 antibodies listed in Table 1 were V-seq containing the amino acid sequence set forth in SEQ ID NO: 10. H Includes. [Table 1]

[0286] Example 2 Generation of CD19-targeted CAR and CAR expression

[0287] Three CD19-targeting CARs, "CAR#2," "CAR#8," and "CAR#15," were generated. The expression of CAR molecules on the cell surface was investigated using a PE-conjugated anti-human LNGFR antibody followed by flow cytometry analysis. LNGFR co-expressed with CAR through the self-cleaving linker 2A sequence, and thus LNGFR expression was used as an indicator of CAR expression. The results are shown in Table 2. [Table 2] The positive control is a CD19-targeted CAR comprising murine scFv SJ25c1 and CD3ζ1XX, referred to as "1928z1XX CAR." Example 3 In vitro activity of CD19-targeted CAR

[0288] The cytotoxicity of these CD19-targeted CARs against CD19-expressing Raji cancer cell lines and CD19 knockout Raji cells was investigated. Each target cell line was seeded into a 384-well plate, and CD19 CAR-T or untransduced T cells (UTD) were added at an effector-to-target (E:T) ratio of 4. Target cell-only wells and effector cell-only wells were included as controls. After 24 hours, cell viability was measured using the CellTiter-Glo® One Solution Assay (Promega, G8462). The percent viability of target cells was calculated by subtracting the signal of the effector cell-only wells from the luminescence signal of the co-culture wells, then dividing by the signal of the target cell-only wells. The percent killing was calculated by subtracting the percent viability of target cells from 100%. As shown in Figures 1A and 1B, T cells containing CAR#2, T cells containing CAR#8, and T cells containing CAR#15 all demonstrated cell-killing activity against Raji cells, in contrast to CD19 knockout Raji cells, indicating that the killing activity of T cells containing these CD19-targeted CARs is antigen-dependent.

[0289] To confirm antigen-dependent cytotoxicity, T cells containing these CD19-targeted CARs were cocultured with CD19-expressing Raji cancer cell lines or CD19 knockout Raji cells. Each target cell line was labeled with cell trace violet dye and then seeded into a 96-well plate. CD19 CAR-T or untransduced T cells (UTD) were added at an effector-to-target (E:T) ratio of 2. Target cell-only wells and effector cell-only wells were included as controls. After 20 hours, cell viability was measured using live / dead dye staining followed by flow cytometry analysis. The percent viability of target cells was calculated by subtracting the signal from the effector cell-only wells from the live cell count measured by flow cytometry, then dividing by the signal from the target cell-only wells. The percent killing was calculated by subtracting the percent viability of target cells from 100%. As shown in Figures 2A and 2B, T cells containing CAR#2, T cells containing CAR#8, and T cells containing CAR#15 all demonstrated cell-killing activity against Raji cells, in contrast to CD19 knockout Raji cells, confirming that the killing activity of T cells containing these CD19-targeted CARs is antigen-dependent.

[0290] Antigen-independent cytokine (IL-2 and IFN-γ) secretion was assessed by co-culture of CAR#2-, CAR#8-, and CAR#15-containing T cells with CD19-expressing Raji cancer cell lines and CD19 knockout Raji cells. Each target cell line was seeded into a 384-well plate, and CD19 CAR-T or untransduced T cells (UTD) were added at an effector-to-target (E:T) ratio of 4. Target cell-only wells and effector cell-only wells were included as controls. After 24 hours, the supernatants and secreted IFN-γ and IL2 were detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). IL-2 secretion results are shown in Figures 3A and 3B, and IFN-γ secretion results are shown in Figures 4A and 4B. T cells containing these CD19-targeted CARs exhibited IL-2 and IFN-γ secretion when co-cultured with Raji cells, in contrast to CD19 knockout Raji cells, indicating antigen-dependent cytokine release of T cells containing these CD19-targeted CARs. Example 4 In vivo activity of CD19-targeted CAR

[0291] The in vivo activity of T cells containing these CD19-targeted CARs was next investigated.

[0292] NOD-scid IL2Rg null NSG mice were transfected with 0.5 × 10 NALM6 cells (a B-ALL cell line) expressing firefly luciferase at wild-type CD19 levels (CD19-WT) or at reduced CD19 levels (CD19-Low, a Nalm6 clone generated by CRISPR-Cas9 editing of the CD19 gene followed by CD19 re-expression using a lentiviral vector). 6 Four days later, CD19-WT (blue, red) and CD19 - Low (green) NALM6-bearing mice were cultured in fresh 5 × 10 4(red) or 2 x 10 5 Mice (blue, green) were treated with a single intravenous injection of #17, #8, #15, or #2 CAR T cells. For a rigorous assessment of CAR T cell efficacy, CAR T cell-treated mice were repeatedly imaged over 60–100 days to assess tumor response and potential for relapse. Tumor burden was analyzed by bioluminescence imaging up to 30 days after T cell infusion and demonstrated for each mouse at the time points shown in Figure 5. #2 CAR T cells demonstrated rapid and effective antitumor activity, resulting in complete tumor eradication of NALM6 WT and NALM6 CD19-Low cells at all T cell doses tested. #17 showed poor tumor control, leading to rapid tumor progression and served as a control. Example 5 #2 scFv binding epitope

[0293] Anti-CD19 antibody binding to the CD19-expressing NALM6 cell line was determined by serial dilution and flow cytometry. Competitive binding of CD19-T2 binders, SJ25c1 (derived from 19-28z CAR) and FMC63 (derived from Kymriah's "Tisagenlecleucel" and Yescarta's "Axicabtagene ciloleucel") scFv, to CD19 was assessed by Biacore SPR. A mixture of 100 nM CD19-HSA-His (Takeda, Cambridge) and increasing concentrations of soluble CD19-T2 SJ25c1 or FMC63 scFv (0, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM) was flowed over a Biacore CMS chip with immobilized CD19-T2 scFv for 3 minutes. Dissociation of the complex bound to the chip was assessed by flowing HBS-EP buffer (300 mM NaCl) for 5 minutes. CD19-HSA-His was purified from HEK293 cells transiently transfected with the pcDNA3.4 plasmid (ThermoFisher), which contains the CD19 extracellular domain fused to a 10-deca-histidine tag, a TEV cleavage site, a GS linker, and a gene expressing HSA. Recombinant CD19-HSA-His10 was purified by nickel-affinity chromatography. The amino acid sequence of CD19-HSA-His10 is shown in Figure 6.

[0294] Epitope binding data for SJ25c, FMC63, and #2 scFv are shown in Figure 7. As shown in Figure 7, #2 scFv competed with FMC63 and SJ25C1 for binding to CD19. These findings confirm that #2 scFv, FMC63, and SJ25C1 scFv bind to overlapping epitopes on CD19. An irrelevant antibody (anti-CTLA4 VHH) that does not bind to CD19 was unable to compete with #2 scFv for binding to CD19. Example 6 Pharmacology Testing summary

[0295] In support of the first-in-human clinical trial, a series of studies were performed to demonstrate improved efficacy of #2 CAR T cells (referred to in this example as "19(T2)28z1XX CAR") compared to 1928z CAR T cells (which had been administered extensively in previous clinical trials), as measured by increased antitumor potency at low T cell doses and increased durability of responses. While CAR T cells engineered to express either CAR exhibited comparable in vitro cytolytic activity, cytokine secretion, and proliferation, #2 CAR T cells achieved greater in vivo efficacy due to their greater functional persistence (Feucht, et al. Nature Medicine (2019);25 (1): 82-88). result

[0296] The in vitro cytotoxic activity of #2 CAR T cells and 1928z-1XX CAR T cells was measured using an 18-hour bioluminescence assay. These two CARs are identical but have different scFvs (#2 vs. SJ25c1). The retroviral vector containing #2 CAR is shown in Figure 8. Recombinant retroviral particles were generated from HEK293 GalV9 packaging cells as previously described (Przybylowski et al., (2006);13(1):95-100). NALM6 cells expressing firefly luciferase (FFL) were used as target cells. T cells were isolated from human PMBCs by negative selection, activated with CD3 / CD28 antibodies bound to magnetic beads for 48 hours, and transduced with a γ-retroviral vector expressing the CAR after bead removal. The CAR+ population was determined by detecting LNGFR, which was coexpressed with the CAR gene. CAR+ T cells were incubated with target NALM6 cells at different effector (E) to target (T) ratios for 18 hours, followed by quantification of FFL activity. The cytotoxic activity of #2 CAR T cells was measured using CD19 +The cytotoxic activity of these CAR T cells was compared with that of 1928z-1xx CAR T cells, which were previously shown to effectively kill NALM6 cells in vitro (Feucht et al., Nature Medicine (2019);25 (1): 82-88). Untransduced (UT) T cells were used as a negative control. NALM6-CD19KO, a cell line generated by CRISPR-Cas9 editing of the CD19 gene (Hamieh et al., Nature (2019);568 (7750): 112-116), was used to demonstrate the specificity of the CAR T cell cytotoxic activity. A representative experiment is shown (n=2 independent experiments on two healthy donors). As shown in Figure 10, #2 CAR T cells and 1928z-1XX CAR T cells exhibited comparable cytotoxicity.

[0297] Next, we measured the in vivo activity of #2 CAR T cells (referred to as "19(T2)28z1XX CAR") and 1928z-1XX CAR T cells. NALM6, a human pre-B acute lymphoblastic leukemia tumor cell line expressing normal (wt) or low (low) levels of CD19 antigen, was transduced with firefly luciferase and green fluorescent protein (GFP) (Zhao et al., Cancer Cell (2015);28:415-428). + low (blue) and CD19 + 2 × 10 fresh NALM6-bearing mice were cultured in a 2×10 5 Mice were treated with a single intravenous injection of untransduced T cells, #2 CAR T cells, or 1928z-1XX CAR T cells. Tumor burden in each mouse was monitored for 85 days by bioluminescence imaging (BLI). There were five mice in each group. The response of each mouse is shown in Figure 11. As shown in Figure 11, the tumor eradication kinetics of #2 CAR T cells and 1928z1XX CAR T cells were similar. CAR cDNA was co-expressed with the LNGFR reporter (to monitor T cell transduction). "19(T2)28z1XX CAR" represents "#2 CAR."

[0298] The activity of #2 CAR T cells and 1928z CAR T cells was tested and compared. First, the CAR expression and phenotype of 1928z CAR T cells and #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") were measured. The vector containing 1928z CAR is shown in Figure 12. CD4 + T cells and CD8 + T cells were transduced with gamma-retroviral vectors expressing the 1928z CAR or the #2 CAR (referred to as "19(T2)28z1XX CAR") and produced using a process representative of clinical manufacturing. Because the SJ25c1 scFv in SGF-1928z is mouse-derived and the #2 scFv (referred to as "19(T2)scFv") is humanized, CAR surface expression was detected using two different goat antibodies specific for either the mouse (GAM) or human (GAH) Fc portion of the IgG heavy chain. As shown in Figure 13, CD4 + T cells and CD8 + More than 50% of the T cells expressed CAR.

[0299] The phenotype of the CAR T cells was then determined by flow cytometry using antibodies recognizing naive T cell, central memory T cell, effector memory T cell, and effector T cell markers, as well as exhaustion markers (LAG3, PD1, and Tim3). "SFG-T2-1XX-GAH" refers to CD4 / CD8 T cells transduced with a gamma-retroviral vector expressing the 19(T2)28z1XX CAR (referred to as "#2 CAR"), while "SFG-1928z-GAM" is derived from T cells transduced with a gamma-retroviral vector expressing the 1928z CAR. As shown in Figure 14, the phenotypes of the 1928z CAR T cells and #2 CAR T cells were similar. The CD4 / CD8 T cell ratio was similar between 1928z and #2 CAR T cells, and the T cell differentiation status, as determined by CD62L / CD45RA and CD45RA / CCR7 staining, was also comparable between both CAR groups (for CD4 and CD8 T cells). The expression of exhaustion markers PD1, LAG3, and TIM3 did not reveal any significant differences between both CAR T cell groups.

[0300] The in vivo activity of #2 CAR T cells and 1928z CAR T cells was then evaluated and compared. 5 CD19 + Four days after injection of NALM6-FFLuc / GFP cells into the tail vein of mice, they were treated with thawed cryopreserved untransduced or CAR T cells. As shown in Figure 15, #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") provided superior tumor control compared to 1928z CAR T cells. CD19 T cells treated with untransduced or CAR T cells were significantly higher in CD19 T cells than CD19 T cells. + The survival of mice bearing NALM6 is plotted in the Kaplan-Meier curve plot shown in Figure 16. As shown in Figure 16, the survival of mice bearing NALM6 treated with #2 CAR T cells (represented as "19(T2)28z1XX CAR T cells") was significantly higher than that of mice bearing CD19 T cells treated with #2 CAR T cells (represented as "19(T2)28z1XX CAR T cells"). +NALM6-bearing mice were cultured at 1 x 10 cells per mouse. 6 At all CAR T cell doses below 1928z, mice survived longer than those treated with 1928z CAR T cells. All 1928z CAR T cell recipients died by day 25, while only one mouse died in the test group that received #2 CAR T cells (designated "19(T2)28z1XX CAR T cells"). 6 In the study, one mouse died in both the control and test groups.

[0301] #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") and CD19 of 1928z CAR T cells + In vivo persistence in the NALM6 leukemia mouse model was also measured. The persistence of CAR T cells after 17 days of treatment and the number of CAR T cells in the bone marrow (n=5 per group) are shown in Figure 17. The PD1, LAG3, and TIM3 expression percentage of CAR T cells, as well as the T n , T cm , T eff , and T em The percentages are shown in FIG.

[0302] As shown in Figure 17, #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") were readily detected in the bone marrow by day 17 and expressed CD4 + CAR T cells and CD8 +Both CAR T cells and 1928z CAR T cells were included. Consistent with their limited persistence, 1928z CAR T cells were scarce (Zhao et al., Cancer Cell (2015);28:415-428). The phenotypes of the respective persisting T cells detected at day 17 were similar (Tn, Tcm, and Tem), indicating a slightly higher fraction of effector T cells (Teff) in the #2 CAR (referred to as "19(T2)28z1XX CAR") group. The latter expressed more PD1 and comparable levels of LAG3 and TIM3. This observation confirms the greater persistence of CAR T cells expressing the CD28 / CD3z-1XX signaling motif (Feucht et al., Nature Medicine (2019);25 (1):82-88).

[0303] These results confirm the greater potency (higher tumor eradication at four dose levels) of the #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") compared to the 1928z CAR T cells. This study further confirms that the phenotype of the #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") is overall similar to that of the 1928z CAR T cells, consistent with the #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") achieving better tumor control due to their greater persistence compared to the shorter-lived 1928z CAR T cells. Methods and Materials Cytolytic assay

[0304] A luciferase-based assay was used to measure the cytotoxicity of T cells transduced with different CAR constructs. NALM6 cells expressing FFLuc-GFP served as target cells. Effector and tumor target cells were co-cultured in triplicate in black-walled 96-well plates at the indicated effector / target ratios. 5 × 10 target cells 4Cells were seeded in a total volume of 100 μL per well with X-VIVO15 supplemented with 5% human AB serum (Gemini), 10 U / ml IL7, and 10 U / ml IL15 (PeproTech, Inc.). Maximum luciferase expression (relative light units (RLU)) was determined using target cells alone plated at the same cell density. After 18 hours of coculture, 100 μL of luciferase substrate (Bright-Glo; Promega) was added directly to each well. Emitted light was determined using a luminescence plate reader. Tumor cell lysis was calculated as (1 - (RLU sample) / (RLU max)) × 100. Isolation, transduction, and expansion of genetically modified human T cells

[0305] Buffy coats from de-identified healthy donors were purchased from the New York Blood Center (institutional review committee exempt). All blood samples were handled in accordance with required ethical and safety procedures. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and activated with Dynabeads® ClinExVivo™ CD3 / CD28 antibody beads, followed by transduction with gamma-retroviral vectors by centrifugation on RetroNectin-coated plates (Takara Bio Inc.) as previously described (Zhao et al., Cancer Cell (2015);28:415-428). After 2 days, activated T cells were subjected to bead removal and transduction by spinoculation as previously described (Hollyman et al., J. Immunotherapy (2009);32 (2):160-180). Transduced cells were then expanded in G-Rex 6M-well plates according to the manufacturer's instructions (Wilson Wolf Corporation). Transduction efficiency was determined 5 days after transduction. CAR-T cells were used fresh or thawed from cryopreservation in 50% CS10 and 5% HSA in PlasmaLyte. Mouse systemic tumor model

[0306] Six- to 12-week-old NOD / SCID / IL-2Rγ null male mice were used under a protocol approved by the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee. All relevant animal use guidelines and ethical regulations were followed. Generally, mice were inoculated with 0.5 × 10 6 NALM6(CD19 + (wt or low expression)-FFLuc-GFP tumor cells were inoculated via tail vein injection, followed by treatment with CAR-T cells at a specific dose for each mouse 4 days after tumor injection. NALM6 cells were delivered at equal tumor burdens, and no mice were excluded prior to CAR-T cell treatment. No randomization or blinding methods were used. Bioluminescence imaging

[0307] Tumor burden was assessed as previously described (Gade et al., Cancer Research (2005);65(16):9080-9088). Briefly, mice were anesthetized with 2% isoflurane for 10 minutes and then intraperitoneally injected with D-luciferin (Xenogen, 3 mg per mouse) resuspended in PBS before imaging. Bioluminescence imaging of injected tumors was performed using an IVIS Imaging System (PerkinElmer). Data were analyzed using Living Image software (PerkinElmer). Antibodies used for flow cytometry

[0308] 1×10 6Seventeen days after treatment with CAR T cells, surface marker expression from CAR T cells generated ex vivo or isolated from mouse bone marrow and spleen was detected using antibodies conjugated with the following fluorophores: APC-Cy7 anti-human CD8 (SK1), APC-Cy7 mouse anti-human CD45 (2D1), BUV395 mouse anti-human CD4 (SK3), BV421 mouse anti-human CD62L (DREG-56), BV650 mouse anti-human CD45RA (HI100), BV480 or BV510 mouse anti-human PD-1 (EH12.1), and BUV737 mouse anti-human CD19 (SJ25C1) (BD Bioscience); PE-Cy7 anti-human CD8 (SK1) and PerCP-eFluor 710 anti-human LAG-3 (3DS223H) (eBioscience); PerCP anti-human CD45RA (HI100), Brilliant Violet785 anti-human Tim-3 (F38-2E2) and PE anti-human CD127 (IL7Ra) (Biolegend). Expression of 1928z CAR was detected by using Alexa Fluor 647 goat anti-mouse AffiniPure IgG, F(ab')2 fragment. Alexa Fluor 647-goat anti-human IgG, F(ab')2 fragment was used to detect #2 CAR (referred to as "19(T2)28z1XX CAR"). 7-AAD (Beckman Coulter) was used as a viability dye. Flow cytometry was performed on a Cytek Aurora instrument (Cytek Biosciences). Data were analyzed using FlowJo software v.10.1 (FlowJo LLC). statistical analysis

[0309] All statistical analyses were performed using Prism 8 (GraphPad) software. Statistical comparisons between two groups were determined by unpaired two-tailed t-test. For in vivo studies, Kaplan-Meier curves were used to present overall survival. Statistical significance was defined as a p-value <0.05. Example 7 Toxicity evaluation summary

[0310] The in vivo toxicity of #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") was evaluated in NSG mice bearing NALM6 leukemia. Mice injected iv with 1928z CAR T cells at the same dose served as controls. Parameters recorded included total body weight, tumor burden, and survival for all mice. A more extensive toxicological investigation focused on the highest dose (1 x 10), a dose that exceeds (10x) the minimum therapeutic (cure) dose for this mouse model and also exceeds (10-100x) the clinically used CAR T cell dose as a percentage of recipient body weight. 6 Mice treated with 10 CAR T cells / recipient were included. These tests included enumeration and phenotyping of T cells at the primary tumor site (bone marrow), serum cytokine measurements (both human, i.e., T cell-derived, and mouse, i.e., host-derived), basic serum biochemistry including liver enzymes, peripheral blood cell counts and organ weights, and comprehensive immunohistochemistry at the end of the study (days 27-28). The infused CAR T cells were produced in a clinically relevant manner, and the two CAR T cell products were comparable.

[0311] Total body weight and survival were not significantly different between the test and control groups. Serum cytokine levels were also comparable, suggesting no CRS in either group. At day 10, ALT and AST enzyme levels were comparable in both test groups. At day 27 / 28, enzyme levels in mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") were lower than those in mice treated with 1928z CAR T cells.

[0312] As expected, #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") outlasted 1928z CAR T cells. Lymphocyte and reticulocyte counts in mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") were higher than in the 1928z CAR T cell group. Mice receiving 1928z CAR T cells exhibited diffuse tumor infiltration, whereas recipients of #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") did not. Mice persisting with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") exhibited mild to moderate allogeneic GVHD and hepatocellular necrosis.

[0313] GVHD is expected in NSG mice treated with high doses of human CAR T cells, although the mechanism of the latter is unclear. Given the heterogeneous nature of the study (prone to heterologous reactivity of human T cells against mouse tissues) and its use with a single donor (whose underlying T cell receptor repertoire is unknown), the significance of this adverse event for autologous treatment in humans is unclear. Evidence for animal models

[0314] The NALM6 model in NSG mice has been used extensively to evaluate the activity of CD19-specific CAR T cells in the Sadelain laboratory (Zhao et al., Cancer Cell (2015);28: 415-428; Feucht, et al. Nature Medicine (2019);25 (1): 82-88; Hamieh et al., Nature (2019);568 (7750): 112-116; Eyquem et al., Nature (2017);543 (7643): 113-117) and others. NALM6 is a human pre-B acute lymphoblastic leukemia (ALL) that expresses CD19 but does not express CD80, CD86, 4-1BBL, or any of the other major costimulatory ligands. Rationale for Dosage and Schedule

[0315] % transduction efficiency and total viable cells based on 1 x 10 6 A single intravenous injection of viable CAR T cells was found to be 10x the minimally effective treatment (1 x 10) in this animal model. 5 The dose was selected considering that it corresponds to an adult CD19 CAR T cell dose (e.g., 0.1-1.5 x 10) when converted by weight from mouse to human. 8 This is approximately 10-100x the approved dose of viable CAR T cells. The suggested starting flat dose for #2 CAR T cells in human clinical trial protocols is 25x10 6 CAR T cells. method Test Articles and Vehicles

[0316] The test articles were 1928z CAR T cells, #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells" in this example), and untransduced T cells prepared by the Cell Therapy and Cell Engineering Facility (CTCEF) at MSKCC. Cells were frozen in liquid nitrogen until the start of the study. On the day of T cell injection, cells were thawed at 37°C and washed with 1% HSA in PlasmaLyte. Cells were then diluted to 1x10 in a volume of 200 μL per mouse using 1% HSA in PlasmaLyte. 6 pieces, 5×10 5 pieces, 2×10 5 pieces, and 1 x 10 5 The number of CAR T cells was calculated based on the total cell count and the CAR T cell dose, which was previously determined using flow cytometry. + Determined by multiplying the percentage. Examination System

[0317] 0.5×10 6Experiments were performed using 6-12 week-old NOD / SCID / IL-2Rγnull male mice injected with 1 × 10 FFLuc-GFP NALM6 cancer cells and 1 × 10 thawed, cryopreserved CD4 / CD8 selected T cells transduced with a γ-retroviral vector containing either the 1928z CAR or the #2 CAR (referred to as "19(T2)28z1XX CAR"). 6 The mice were injected with 100 mg of ribosomal protein (RIP) four days after tumor cell injection. Untreated mice were expected to develop hind limb paralysis due to bone marrow infiltrating leukemia by day 16–18.

[0318] The wild-type NALM6 human tumor cell line is a well-characterized model of pre-B acute lymphoblastic leukemia and has been used by the Sadelain laboratory in numerous peer-reviewed publications (Zhao et al., Cancer Cell (2015);28: 415-428; Feucht, et al. Nature Medicine (2019);25 (1): 82-88; Hamieh et al., Nature (2019);568 (7750): 112-116; Eyquem et al., Nature (2017);543 (7643): 113-117). Test Design

[0319] 115 mice were divided into a 1928z CAR group, a #2 CAR (referred to as "19(T2)28z1XX CAR") group, and an untransduced group. All mice received 0.5 x 10 T cells 4 days before T cell treatment. 6 Mice received FFLuc-GFP NALM6 tumor cells via tail vein injection. On study day 0, mice were assigned to treatment groups. Each group received a single dose (200 μl) of test article administered intravenously. Group assignments are outlined in Table 3. [Table 3] Toxicological parameters

[0320] Toxicity was monitored by changes in body weight after test article administration. 10 and 28 days after test article administration, 1 x 10 1928z CAR T cells and #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") were administered. 6 Blood samples from individual dose groups were collected from the orbital venous plexus after anesthesia under 2% isoflurane. Blood samples were collected into tubes containing serum separator and used to measure cytokine and liver enzyme levels. On days 27 and 28, 1 × 10 1928z CAR and #2 CAR (referred to as "19(T2)28z1XX CAR") were administered. 6 Mice from individual dose groups were subjected to complete necropsy analysis on the days indicated. Cytokine analysis

[0321] Serum was collected from mice at days 10 and 28 after CAR T treatment at 1 × 10 6Blood samples were collected from both the 1928z CAR group and the #2 CAR (referred to as "19(T2)28z1XX CAR") group, which received 1928z CAR T cell doses. Samples were stored at -80°C until analysis. ProcartaPlex assays were used to quantify mouse cytokine (MCP-1, IL-6, and G-CSF) and human cytokine (IL2, IL3, IFN-g, GM-CSF, granzyme B, and TNF-α) levels. All ProcartaPlex-related reagents in this study were purchased from ThermoFisher Scientific. Serum from a mouse model of severe cytokine release syndrome was used as a positive control (Giavridis et al., Nature Medicine (2018);24 (6): 731-738). 60 μL of beads from each cytokine simplex kit was pooled, and 50 μL of the bead mixture was distributed into each well of a 96-well plate according to the manufacturer's protocol. Standards, quality controls, and samples diluted 1:4 using universal assay buffer were added to the wells. The plates were sealed and shaken at 500 rpm for 30 minutes at room temperature and transferred to 4°C. After overnight incubation, the plates were shaken at 500 rpm for an additional 30 minutes at room temperature. The plates were washed, and detection antibodies were added to each well. The plates were sealed and shaken at 500 rpm for 30 minutes at room temperature. After washing, streptavidin-RPE solution was added to each well. The plates were sealed and shaken at 500 rpm for 30 minutes at room temperature. After a final wash step, the beads were resuspended in reading buffer, and assay signals were acquired on a Luminex 200 instrument. Standard curves for individual cytokines were generated using a five-parameter logistic equation, and the concentrations of individual cytokines in each sample were calculated using xPONENT software (version 4.2.1509.0). Statistics

[0322] All statistical analyses were performed using Prism 8 (GraphPad) software. Statistical comparisons between two groups were determined by unpaired two-tailed t-test. Statistical significance was defined as a p-value <0.05. result

[0323] The change in mouse weight during treatment with 1928z CAR T cells and #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") was measured. NALM6 tumor-bearing mice were treated with 1x10 6 pieces, 5×10 5 pieces, 2×10 5 pieces, or 1 x 10 5 1928z CAR T cells or #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") (n=10 per group), while non-transduced T cell treatment served as the control group (n=5). The results are shown in Figure 18. As shown in Figure 18, the change in body weight was similar between the two CAR T cell treatments.

[0324] Next, liver enzymes were measured in the serum of mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") and 1928z CAR T cells. Quantification of liver enzyme levels from two CAR T-treated mice at days 10 and 27 / 28 (dose level = 1 x 10 6 CAR T, n=10 per group) are presented in Figure 19. 6 NALM6 tumor-bearing mice treated with 1928z CAR T cells or #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") were assessed for ALT and AST levels on day 10 and days 27 / 28. As shown in Figure 19, ALT and AST enzyme levels on day 10 were comparable in both test groups. Enzyme levels in mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") on day 27 / 28 were lower than those in mice treated with 1928z CAR T cells.

[0325] 1×10 6Human and mouse cytokine levels were measured on days 10 and 27 / 29 post-infusion in mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") or 1928z CAR T cells. Figure 20 shows quantification of human cytokine levels from serum obtained from two groups of different CAR T-treated mice on days 10 and 27 / 28 (dose level = 1 x 10 6 CAR T cells, n=10 per group). Serum collected from a mouse model of severe cytokine release syndrome was used as a positive control (n=2) (Giavridis et al., Nature Medicine (2018);24 (6): 731-738). IL2, IL3, GM-CSF, granzyme B, and TNF-alpha levels on days 10 and 27 / 28 were comparable between the two CAR T cell groups. IFN-γ levels from the 19(T2)28z1XX CAR T treatment group on days 27 / 28 were higher than those in mice receiving 1928z CAR T cells.

[0326] Figure 21 shows the efficacy and safety of 1928z CAR T cells or #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") on days 10 and 27 / 28 (dose level = 1 x 10 6Figure 1 shows the results of quantification of mouse cytokine levels from serum of mice treated with either 1928z CAR T cells or 1928z CAR T cells (n=10 per group). Serum collected from a mouse model of severe cytokine release syndrome was used as a positive control (n=2) (Giavridis et al., Nature Medicine (2018);24 (6): 731-738). On day 10, mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") had slightly higher levels of MCP-1 and G-CSF than mice infused with 1928z CAR T cells, while IL6 levels were comparable between both CAR T cell groups. On day 27 / 28, mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") had levels of MCP-1, IL-6, and G-CSF similar to those in the 1928z CAR T group. These findings suggest that there was no evidence of cytokine release syndrome (CRS) in either group.

[0327] Blood cell counts were measured on days 27 / 28 post-infusion in mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") and mice treated with 1928z CAR T cells. Two different CAR T treatment groups (dose level = 1x10 6 Quantification of essential blood cells at day 27 / 28 for the two CAR T groups (1×10 CAR T, n=10 per group) is presented in Figure 22. Comparison of neutrophil, lymphocyte, and monocyte counts, as well as reticulocyte counts, is shown. As shown in Figure 22, neutrophils and monocytes were significantly higher in the two CAR T groups (1×10 CAR T, n=10 per group). 6 Mice in the 19(T2)28z1XX CAR T-cell treated group had higher lymphocyte and reticulocyte counts than the 1928z CAR T-cell group.

[0328] Additionally, body and organ weights were measured at necropsy for mice treated with #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") and mice treated with 1928z CAR T cells. Comparison of body and essential organ weights from the two different CAR T treatment groups on day 27 / 28 (dose level = 1x10 6 CAR T). The results are shown in Figure 23. As shown in Figure 23, body weight, spleen weight, and kidney weight were comparable between the two treatment groups 1 and 2. The liver weight of mice in the 19(T2)28z1XX CAR T treatment group (denoted as "SFG-19(T2)28z-1XX") was lighter than that of mice in the 1928z CAR T group (denoted as "SFG-1928z"). The difference in heart weight was attributed to unequal technical handling.

[0329] #2 CD3 in the liver, bone marrow, and spleen of mice treated with CAR T cells (referred to as "19(T2)28z1XX CAR T cells") and mice treated with 1928z CAR T cells + and CD19 + Immunohistochemistry was performed to assess lymphocyte and tumor infiltration levels in the liver, spleen, and bone marrow from mice 27 / 28 days after CAR T treatment (dose level = 1 × 10 6 CAR T, n=6) are shown in Figure 24. As shown in Figure 24, mice in the 19(T2)28z1XX CAR T treatment group (denoted "SFG-19(T2)28z-1XX") had higher levels of CD3+ cells in all of the organs evaluated than animals treated with 1928z CAR T cells (denoted "SFG-1928z"). Tumor infiltration was observed in all of the organs tested from the 1928z CAR T group, but not in the 19(T2)28z1XX CAR T group. conclusion

[0330] The high dose is 1 × 10 per recipient 6The toxicity profile of #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") was compared with that of 1928z CAR T cells in 1928z CAR T cells. No differences in total body weight or survival were observed. Serum cytokine levels measured on day 10 were comparable between the two groups, suggesting that #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") are not more likely to induce cytokine release syndrome (CRS) than 1928z CAR T cells. Liver enzymes were elevated in both groups on days 10 and 17, but to a lesser extent in recipients of #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") than in the group receiving 1928z CAR T cells. Peripheral blood cell counts showed higher lymphocyte counts in recipients of #2 CAR T cells (designated "19(T2)28z1XX CAR T cells"), consistent with greater persistence of #2 CAR T cells, as demonstrated by FACs analysis (day 17) and tissue IHC (day 27 / 28). Isolated reticulocytosis without anemia, erythrocytosis, or erythroid hyperplasia was observed and therefore was not considered an adverse event.

[0331] Organ weights were similar between the two groups, except for the liver, which was heavier in mice treated with 1928z CAR T cells due to substantial tumor progression (BLI and IHC). In contrast, IHC demonstrated the absence of tumors in mice treated with #2 CAR T cells (designated "19(T2)28z1XX CAR T cells"), confirming the BLI findings in these same mice. Multiorgan lymphocytic infiltration was observed in #2 CAR T cell (designated "19(T2)28z1XX CAR T cells") recipient mice, consistent with xenograft-versus-host disease (GVHD). The latter was confirmed by the finding of epithelial necrosis in these tissues. Mild to moderate hepatocellular necrosis was also observed in mice injected with #2 CAR T cells (designated "19(T2)28z1XX CAR T cells"). This microscopic finding is not commonly associated with GVHD and therefore represents a possible adverse event. The impact of this finding on liver function was limited, given the modest elevation of liver enzymes (AST only, not ALT) and normal bilirubin levels. Other pathological findings were minor and equally represented between the two treatment groups and therefore were not considered to represent significant findings. T cell flow cytometry profiles were similar between the two treatment groups, consistent with the expected finding that #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") exhibited increased therapeutic efficacy due to greater CAR T cell persistence.

[0332] Toxicity studies evaluating the #2 CAR T cells (designated "19(T2)28z1XX CAR T cells") compared to the clinically tested 1928z CAR T cells (Park et al., N. Engl. J. Med. (2018);378 (5): 449-459) revealed mild to moderate GVHD as well as mild to moderate hepatocellular necrosis in mice treated with the #2 CAR T cells (designated "19(T2)28z1XX CAR T cells").

[0333] From the data presented in this Example and Example 6, it can be concluded that #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") are more potent due to their greater persistence compared to 1928z CAR T cells, but do not pose greater risks. The relatively increased potency of #2 CAR T cells (referred to as "19(T2)28z1XX CAR T cells") allows for the administration of 25x10 CAR T cells, a relatively lower dose than the majority of CAR T cell trials targeting CD19. 6 Human clinical trial designs starting with CAR T cell doses are supported. Example 8 Pharmacology Testing On-cell binding of anti-CD19 antibodies

[0334] V of the scFv of the present disclosure H and V L On-cell binding of anti-CD19 antibodies, including (I) and (II), was assessed by flow cytometry on Raji and NALM-6 cell lines, which express endogenous CD19. Each antibody was tested in both the CD19-positive parental line and the corresponding CD19 knockout Raji and NALM-6 lines to confirm on-cell target-specific binding. The flow cytometry chromatograms shown in Figure 25 demonstrate a 1-2 log shift in the CD19-positive line relative to background binding in the corresponding knockout cell line.

[0335] Flow cytometry results of serial dilutions shown in Figures 26A-26C demonstrate saturable binding to NALM-6 cells. EC50 values ​​calculated from the curves are presented in Table 4. Exemplary anti-CD19 antibodies bind to CD19-positive NALM-6 cells with high affinity, starting from at least 0.2 nM. The higher affinity observed for CD19-positive cells compared to the soluble protein suggests that these antibodies bind to epitopes more naturally present on cells than are present in the CD19-HSA fusion protein. All of the anti-CD19 antibodies shown in Table 4 are V-seq antibodies containing the amino acid sequence set forth in SEQ ID NO: 10.H Includes. [Table 4]

[0336] Example 9 Phase I Study of CD19-Targeted 19(T2)28z1XX CAR T Cells for Adult Patients with Relapsed or Refractory B-Cell Malignancies

[0337] This example describes a phase I trial of CD19-targeted 19(T2)28z1XX CAR T cells in adult patients with relapsed or refractory B-cell malignancies. "19(T2)28z1XX" and "19(T2)28z1XX" are used interchangeably herein. Autologous CAR T-cell therapy targeting CD19, a B-cell-specific surface antigen, has demonstrated favorable clinical responses for relapsed or refractory (R / R) B-cell lymphoma (BCL). However, despite initial complete response (CR) rates of 40-60%, only a small proportion of patients achieve long-lasting remissions, necessitating further improvements in the efficacy of CAR therapy by preventing relapse and achieving deeper clinical responses (CR).

[0338] Study Design and Methods: This study is a single-center Phase I clinical trial of 19(T2)28z1XX in patients with R / RB cell malignancies. Key disease eligibility criteria include R / R diffuse large B-cell lymphoma (DLBCL), high-grade BCL, primary mediastinal BCL, indolent BCL, and chronic lymphocytic leukemia (CLL). Patients with a history of CD19 CAR therapy are eligible as long as CD19 expression is confirmed. Key exclusion criteria include ongoing immunosuppression, e.g., systemic GvHD treatment, and active CNS disease.

[0339] This study will use a 3+3 dose escalation design to identify the maximum tolerated dose for BCL. Five flat dose levels are planned: 25 x 10 6 pieces, 50×10 6 pieces, 100×10 6 pieces, 150×10 6pieces, and 200 x 10 6 CAR T cells. Patients will receive conditioning chemotherapy consisting of three days of fludarabine and cyclophosphamide, followed by a single infusion of 19(T2)28z1XX CAR T cells. Patients with DLBCL, high-grade BCL, and primary mediastinal BCL will be eligible to participate in the dose-escalation phase. Once the recommended phase 2 dose (RP2D) is determined, the study will begin a dose-expansion phase with two cohorts. Cohort 1 will include patients with DLBCL, high-grade BCL, and primary mediastinal BCL (i.e., same eligibility criteria as the dose-escalation phase). Cohort 2 will include patients with indolent BCL, CLL, and Richter's transformation. The dose-expansion portion of the study is designed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CARs for multiple indications. Up to 60 patients will be enrolled.

[0340] The primary objective of this study is to evaluate the safety and tolerability of 19(T2)28z1XX and determine the recommended Phase 2 dose. Key secondary objectives include evaluating the efficacy and pharmacokinetics of 19(T2)28z1XX. Exploratory objectives include assessment of B-cell aplasia and measurable residual disease (MRD), characterization of 19(T2)28z1XX CAR T-cell phenotype before and after infusion, and analysis of serum cytokines.

[0341] While the subject matter and certain advantages of the present disclosure have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not limited to the particular embodiments of the processes, machines, manufactures, and compositions, and methods described herein. As will be readily apparent to those skilled in the art from the disclosure of the subject matter of the present disclosure, any currently existing or later developed process, machine, manufacture, composition, or method that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein can be utilized in accordance with the subject matter of the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions, or methods.

[0342] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the entire disclosures of which are incorporated herein by reference for all purposes.

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that specifically binds to CD19, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain is a) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; and b) i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; iii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21; iv) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57; v) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59; vi) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61; vii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63; or viii) a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 66; Including, CAR.

2. The extracellular antigen-binding domain may be a single-chain variable fragment (scFv), Fab, or F(ab). ２ The CAR of claim 1, comprising:

3. The CAR of claim 2, wherein the extracellular antigen-binding domain comprises an scFv.

4. The CAR of claim 3, wherein the scFv is a human scFv.

5. The CAR according to any one of claims 1 to 4, wherein the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

9.

6. the light chain variable region (a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21 The CAR according to any one of claims 1 to 5, comprising:

7. (a) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13; (b) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:17; or (c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21; A CAR according to any one of claims 1 to 6.

8. The CAR of any one of claims 1 to 7, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:

10.

9. The CAR of any one of claims 1 to 8, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

10.

10. The CAR of any one of claims 1 to 9, wherein the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO:

67.

11. The CAR of any one of claims 1 to 10, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO:

67.

12. The CAR of any one of claims 1 to 11, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, or SEQ ID NO:

22.

13. The CAR of any one of claims 1 to 12, wherein (a) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 10; and (b) the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO:

67.

14. The CAR of any one of claims 1 to 13, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, or SEQ ID NO:

67.

15. The CAR of any one of claims 1 to 14, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 18, or SEQ ID NO:

22.

16. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18; (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 22; (d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58; (e) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60; (f) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62; (g) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64; or (h) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

67. A CAR according to any one of claims 1 to 15.

17. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; (b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18; or (c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 22; A CAR according to any one of claims 1 to 16.

18. The CAR of any one of claims 1 to 17, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region.

19. The CAR according to claim 18, wherein the linker consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

20. The heavy chain variable region and the light chain variable region are, from N-terminus to C-terminus, V Ｌ -V Ｈ The CAR according to any one of claims 1 to 19, wherein the CAR is located at

21. The CAR of any one of claims 1 to 20, wherein the extracellular antigen-binding domain comprises an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 23, or is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO:

23.

22. The CAR of any one of claims 1 to 21, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide.

23. The CAR of claim 22, wherein the transmembrane domain comprises a CD28 polypeptide.

24. The CAR of any one of claims 1 to 23, wherein the intracellular signaling domain comprises a CD3ζ polypeptide.

25. The CAR of claim 24, wherein the CD3ζ polypeptide is a modified CD3ζ polypeptide.

26. The CAR of claim 25, wherein the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 consisting of two loss-of-function mutations.

27. The CAR of claim 26, wherein the native ITAM1 consists of the amino acid sequence set forth in SEQ ID NO:

31.

28. The CAR of claim 26, wherein the ITAM2 variant consists of the amino acid sequence set forth in SEQ ID NO:

37.

29. The CAR of claim 26, wherein the ITAM3 variant consists of the amino acid sequence set forth in SEQ ID NO:

41.

30. The CAR of any one of claims 25 to 29, wherein the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:

43.

31. 31. The CAR of any one of claims 1 to 30, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region.

32. The CAR of claim 31, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

33. The CAR of claim 32, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide.

34. 34. The CAR of any one of claims 1 to 33, which is expressed from a vector.

35. The CAR of claim 34, wherein the vector is a retroviral vector.

36. A cell comprising the CAR of any one of claims 1 to 35.

37. The cell of claim 36, wherein the CAR is transduced.

38. 38. The cell of claim 36 or claim 37, wherein the CAR is constitutively expressed on the surface of the cell.

39. 39. The cell of any one of claims 36 to 38, which is an immunoresponsive cell.

40. 40. A cell according to any one of claims 36 to 39, which is a cell of the lymphoid or myeloid lineage.

41. 41. The cell of any one of claims 36 to 40, selected from the group consisting of T cells, natural killer (NK) cells, stem cells capable of differentiating into lymphoid cells, and stem cells capable of differentiating into myeloid cells.

42. 42. The cell of any one of claims 36 to 41, which is a T cell.

43. 43. The cell of claim 41 or claim 42, wherein the T cell is selected from the group consisting of helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells.

44. 42. The cell of any one of claims 36 to 41, which is a NK cell.

45. 45. The cell of claim 44, wherein the NK cell is derived from a stem cell.

46. 46. ​​The cell of claim 41 or 45, wherein the stem cell is a pluripotent stem cell.

47. 47. The cell of claim 46, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

48. A nucleic acid molecule encoding the CAR of any one of claims 1 to 35.

49. 49. The nucleic acid molecule of Claim 48, further comprising a promoter operably linked to the CAR.

50. 50. The nucleic acid molecule of claim 49, wherein the promoter is endogenous or exogenous.

51. 51. The nucleic acid molecule of claim 50, wherein the exogenous promoter is selected from the group consisting of an elongation factor (EF)-1 promoter, a cytomegalovirus immediate early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, a metallothionein promoter, and a ubiquitin C promoter.

52. 50. The nucleic acid molecule of claim 49, wherein the promoter is an inducible promoter.

53. 53. The nucleic acid molecule of claim 52, wherein the inducible promoter is selected from the group consisting of a NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL-2 promoter, a 4-1BB promoter, a PD1 promoter, and a LAG3 promoter.

54. 51. The nucleic acid molecule of claim 50, wherein the promoter is an endogenous promoter.

55. 55. The nucleic acid molecule of claim 54, wherein the endogenous promoter is selected from a TCR alpha promoter, a TCR beta promoter, and a beta2-microglobulin promoter.

56. 56. A vector comprising a nucleic acid molecule according to any one of claims 48 to 55.

57. 57. The vector of claim 56, which is a retroviral vector.

58. 56. A cell expressing a nucleic acid molecule according to any one of claims 48 to 55.

59. 59. The cell of claim 58, which is a T cell or a natural killer (NK) cell.

60. 60. A composition comprising a cell according to any one of claims 36 to 47, 58 and 59.

61. 61. The composition of claim 60, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

62. 1 x 10 ６ Pieces to 5 x 10 ８ 62. The composition of claim 60 or claim 61, comprising cells between .

63. 1 x 10 ６ Pieces to 1 x 10 ８ 62. The composition of claim 60 or claim 61, comprising cells between .

64. 1 x 10 ６ Pieces to 5 x 10 ７ 62. The composition of claim 60 or claim 61, comprising cells between .

65. 2.5 x 10 ７ 65. The composition of any one of claims 60 to 64, comprising cells. thing.

66. 66. A composition for use in a method for reducing tumor burden in a subject, the composition comprising a cell described in any one of claims 36 to 47, claims 58 and 59 or a composition described in any one of claims 60 to 65.

67. 67. The composition of claim 66, wherein the method reduces the number of tumor cells, shrinks tumor size, and / or eradicates tumors in the subject.

68. A composition for increasing or prolonging the survival of a subject having a neoplasm, the composition comprising a cell described in any one of claims 36 to 47, claims 58 and 59 or a composition described in any one of claims 60 to 65.

69. A composition for use in a method for treating and / or preventing a neoplasm in a subject, the composition comprising a cell described in any one of claims 36 to 47, claims 58 and 59 or a composition described in any one of claims 60 to 65.

70. 70. The composition of any one of claims 66 to 69, wherein the tumor and / or neoplasm is CD19 associated.

71. 71. The composition of any one of claims 66 to 70, wherein the tumor and / or neoplasm is a blood cancer.

72. 72. The composition of claim 71, wherein the hematological cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma.

73. 73. The composition of claim 72, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia.

74. 73. The composition of claim 72, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

75. 71. The composition of any one of claims 66 to 70, wherein the tumor and / or neoplasm is a B-cell malignancy.

76. 76. The composition of claim 75, wherein the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma.

77. 77. The composition of any one of claims 66 to 76, wherein the tumor and / or neoplasm is a B-cell lymphoma.

78. 78. The composition of claim 77, wherein the B-cell lymphoma is relapsed or refractory (R / R) B-cell lymphoma.

79. 79. The composition of any one of claims 66 to 78, wherein the subject is a human subject.

80. 66. A cell according to any one of claims 36 to 47, 58 and 59 or a composition according to any one of claims 60 to 65 for use in therapy.

81. A cell according to any one of claims 36 to 47, claims 58 and 59 or a composition according to any one of claims 60 to 65 for use in the treatment and / or prevention of a neoplasm in a subject.

82. A cell according to any one of claims 36 to 47, claims 58 and 59 or a composition according to any one of claims 60 to 65 for use in increasing or prolonging the survival of a subject having a neoplasm.

83. A cell according to any one of claims 36 to 47, claims 58 and 59 or a composition according to any one of claims 60 to 65 for use in the treatment and / or prevention of a neoplasm in a subject.

84. 84. A cell or composition for use according to any one of claims 81 to 83, wherein the tumor and / or neoplasm is CD19 associated.

85. 85. The cell or composition for use according to any one of claims 81 to 84, wherein the tumor and / or neoplasm is a blood cancer.

86. 86. The cell or composition for use of claim 85, wherein the hematological cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma.

87. 87. The cell or composition for use of claim 86, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia.

88. 87. The cell or composition for use of claim 86, wherein the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

89. 85. The cell or composition for use according to any one of claims 81 to 84, wherein the tumor and / or neoplasm is a B-cell malignancy.

90. 90. The cell or composition for use of claim 89, wherein the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma.

91. 91. The cell or composition for use according to any one of claims 81 to 90, wherein the tumor and / or neoplasm is a B-cell lymphoma.

92. 92. The cell or composition for use of claim 91, wherein the B cell lymphoma is relapsed or refractory (R / R) B cell lymphoma.

93. 93. The cell or composition for use according to any one of claims 81 to 92, wherein the subject is a human subject.

94. 40. A method for producing a cell comprising the CAR of any one of claims 1 to 35, the method comprising the step of introducing into the cell a nucleic acid molecule encoding the CAR of any one of claims 1 to 35.

95. 60. A kit for reducing tumor burden in a subject, for treating and / or preventing a neoplasm in a subject, and / or for increasing or prolonging survival of a subject having a neoplasm, the kit comprising a cell described in any one of claims 36 to 47, claims 58 and 59.

96. 96. The kit of claim 95, further comprising instructions for using the cells to reduce tumor burden in a subject, to treat and / or prevent a neoplasm in a subject, and / or to increase or prolong survival of a subject having a neoplasm.

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