Prostate-specific membrane antigen car and use methods thereof

Abstract

To provide modified immune cells (e.g., modified T cells) comprising a chimeric antigen receptor (CAR) having affinity to prostate-specific membrane antigen (PSMA) (e.g., human PSMA).SOLUTION: The disclosure provides a modified immune cell, e.g., a modified T cell, comprising a CAR having affinity to PSMA, and a dominant-negative receptor and / or a switch receptor. The disclosure also provides a modified immune cell, e.g., a modified T cell, comprising a CAR having affinity to PSMA, and a dominant-negative receptor and / or a switch receptor, where the modified cell is capable of expressing and secreting a bispecific antibody.SELECTED DRAWING: Figure 1B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 365(b) (35 U.S.C. § 119(e)) to international application PCT / US2019 / 020729, filed March 5, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Breaking tolerance to self-antigens poses a major challenge to the application of immunotherapy to solid malignancies. Vaccine strategies that aim to harness endogenous antitumor T cells are limited by the T cell receptor (TCR) repertoire and can be eliminated in the thymus as part of central tolerance or rendered nonfunctional by postthymic mechanisms of peripheral tolerance. One strategy to overcome such obstacles is to generate genetically engineered T cells that are redirected to tumor antigens using a chimeric antigen receptor (CAR) approach. CAR T cells use genetically programmed patient-derived lymphocytes transduced with a chimeric receptor gene to combine the antigen-recognition domain of a specific antibody with the signaling domain of a TCR.

[0003] Prostate-specific membrane antigen (PSMA) is a membrane-bound protein expressed on the cell surface and has been reported to be highly overexpressed in prostate cancer tissues. PSMA expression directly correlates with tumor grade and stage progression and is thought to confer a selective growth advantage to prostate cancer cells. Thus, PSMA may be an ideal target for immunotherapy against prostate cancer.

[0004] Another major challenge in cancer immunotherapy is the hostile microenvironment in which the target tumor resides. For example, immunoinhibitory receptor ligands, such as PDL1 (CD274) that binds to PD1 (CD279), are upregulated in the tumor microenvironment and negatively regulate T cell activity. In addition, TGF-β, which is overexpressed in prostate tumor cells, can act as an immunosuppressive molecule.

[0005] Thus, there is a need in the art for novel cancer immunotherapies that target PSMA. The present invention fulfills this need. Summary of the Invention

[0006] The present invention is based on the discovery that humanized prostate-specific membrane antigen (PSMA) chimeric antigen receptor (CAR) T cells exhibit potent antitumor activity. The present invention is also based on the discovery that PSMA-CAR T cells containing dominant-negative receptors and / or switch receptors exhibit significantly enhanced antitumor activity.

[0007] Thus, in certain aspects, the present disclosure provides an engineered immune cell or a precursor thereof comprising: (a) a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 183; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 184; and (b) a dominant-negative receptor and / or a switch receptor.

[0008] In certain exemplary embodiments, the VH comprises the sequence of SEQ ID NO: 191. In certain exemplary embodiments, the VL comprises the sequence of SEQ ID NO: 192.

[0009] In certain exemplary embodiments, the antigen-binding domain comprises an antibody or antigen-binding fragment thereof, hi certain exemplary embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0010] In certain exemplary embodiments, the transmembrane domain comprises a transmembrane region derived from CD8. In certain exemplary embodiments, the transmembrane region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO:88. In certain exemplary embodiments, the transmembrane domain further comprises a hinge region derived from CD8. In certain exemplary embodiments, the hinge region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO:86.

[0011] In certain exemplary embodiments, the intracellular domain comprises a 4-1BB signaling domain and a CD3 zeta signaling domain. In certain exemplary embodiments, the intracellular domain comprises an ICOS signaling domain and a CD3 zeta signaling domain. In certain exemplary embodiments, the intracellular domain comprises a variant ICOS signaling domain and a CD3 zeta signaling domain. In certain exemplary embodiments, the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO:92. In certain exemplary embodiments, the ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID NO:203. In certain exemplary embodiments, the variant ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID NO:95. In certain exemplary embodiments, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO:97 or 100.

[0012] In certain exemplary embodiments, the dominant negative receptor is a truncated variant of a wild-type protein associated with negative signaling, hi certain exemplary embodiments, the truncated variant of a wild-type protein associated with negative signaling comprises the amino acid sequence set forth in SEQ ID NO:115.

[0013] In certain exemplary embodiments, the switch receptor comprises a first domain derived from a first polypeptide associated with a negative signal; and a second domain derived from a second polypeptide associated with a positive signal. In certain exemplary embodiments, the first domain comprises at least a portion of the extracellular domain of the first polypeptide associated with a negative signal, and the second domain comprises at least a portion of the intracellular domain of the second polypeptide associated with a positive signal. In certain exemplary embodiments, the switch receptor further comprises a transmembrane domain of the switch receptor. In certain exemplary embodiments, the transmembrane domain of the switch receptor comprises the transmembrane domain of the first polypeptide associated with a negative signal; or the transmembrane domain of the second polypeptide associated with a positive signal.

[0014] In certain exemplary embodiments, the first polypeptide associated with negative signaling is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR. In certain exemplary embodiments, the second polypeptide associated with positive signaling is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R.

[0015] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of CD28; and a second domain comprising at least a portion of the intracellular domain of CD28. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:117.

[0016] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of PD1; and a second domain comprising at least a portion of the intracellular domain of CD28. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:119.

[0017] In certain exemplary embodiments, the first domain comprises at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:121.

[0018] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1, the extracellular domain comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of CD28. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:121.

[0019] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1, the extracellular domain comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of 4-1BB. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:215.

[0020] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TIM-3; and a second domain comprising at least a portion of the intracellular domain of CD28. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:127.

[0021] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rα1. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:123.

[0022] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rβ1. In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:125.

[0023] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a dominant-negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

[0024] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising: a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 213 or 215.

[0025] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising: a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 117 or 119.

[0026] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising: a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 121.

[0027] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising: a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 127.

[0028] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising: a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 123.

[0029] In another aspect, the present disclosure provides an engineered immune cell or a precursor thereof, comprising: a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 125.

[0030] In certain exemplary embodiments, the modified cells are modified T cells. In certain exemplary embodiments, the modified T cells are autologous cells. In certain exemplary embodiments, the modified T cells are allogeneic cells. In certain exemplary embodiments, the modified cells are cytotoxic T lymphocytes (CTLs). In certain exemplary embodiments, the modified cells are derived from human cells.

[0031] In another aspect, the present disclosure provides an isolated nucleic acid comprising: (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 183; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 184; and (b) a second nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor.

[0032] In certain exemplary embodiments, the VH comprises the sequence of SEQ ID NO: 191. In certain exemplary embodiments, the VL comprises the sequence of SEQ ID NO: 192.

[0033] In certain exemplary embodiments, the first nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 246, 248, 250, 252, 254, or 256. In certain exemplary embodiments, the second nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216.

[0034] In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker. In certain exemplary embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). In certain exemplary embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. In certain exemplary embodiments, the self-cleaving peptide is a 2A peptide. In certain exemplary embodiments, the 2A peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A). In certain exemplary embodiments, the 2A peptide is T2A. In certain exemplary embodiments, the 2A peptide is F2A.

[0035] In certain exemplary embodiments, the isolated nucleic acid comprises, in a 5' to 3' direction: a first nucleic acid sequence, a linker, and a second nucleic acid sequence.

[0036] In certain exemplary embodiments, the isolated nucleic acid comprises, in a 5' to 3' direction: a second nucleic acid sequence, a linker, and a first nucleic acid sequence.

[0037] In another aspect, the present disclosure provides an isolated nucleic acid comprising: a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), the first nucleic acid sequence comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 192; and a second nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor, the second nucleic acid sequence comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216.

[0038] In another aspect, the present disclosure provides an isolated nucleic acid comprising: a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 192; and a second nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor comprising the nucleic acid sequence set forth in SEQ ID NO: 116.

[0039] In certain exemplary embodiments, the first and second nucleic acid sequences are separated by a linker comprising a nucleic acid sequence encoding T2A, hi certain exemplary embodiments, the first and second nucleic acid sequences are separated by a linker comprising a nucleic acid sequence encoding F2A.

[0040] In another aspect, the disclosure provides an expression construct comprising the aforementioned isolated nucleic acid.

[0041] In certain exemplary embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In certain exemplary embodiments, the expression construct is a lentiviral vector. In certain exemplary embodiments, the lentiviral vector further comprises an EF-la promoter. In certain exemplary embodiments, the lentiviral vector further comprises a rev response element (RRE). In certain exemplary embodiments, the lentiviral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain exemplary embodiments, the lentiviral vector further comprises a cPPT sequence.

[0042] In certain exemplary embodiments, the lentiviral vector further comprises an EF-la promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence.

[0043] In certain exemplary aspects, the lentiviral vector is a self-inactivating lentiviral vector.

[0044] In another aspect, the present disclosure provides a method for generating the aforementioned modified immune cells or precursors thereof, comprising introducing one or more of the aforementioned nucleic acids or the aforementioned expression constructs into an immune cell.

[0045] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune cells described above.

[0046] In certain exemplary embodiments, the method further comprises administering lymphodepleting chemotherapy to the subject. In certain exemplary embodiments, the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and / or fludarabine.

[0047] In another aspect, the present disclosure provides a method of treating prostate cancer in a subject in need thereof, the method comprising: administering to the subject lymphodepleting chemotherapy, comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and administering to the subject modified T cells, wherein the modified T cells comprise a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell; and a dominant-negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 192.

[0048] In another aspect, the present disclosure provides a method of treating metastatic castration-resistant prostate cancer in a subject in need thereof, the method comprising: administering to the subject lymphodepleting chemotherapy, comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and administering to the subject engineered T cells, wherein the engineered T cells comprise a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell; and a dominant-negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 192.

[0049] [The present invention 1001] (a) a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 183; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 184; (b) dominant-negative receptors and / or switch receptors A modified immune cell or a precursor thereof, comprising: [The present invention 1002] 1001. The modified cell of the present invention, wherein VH comprises the sequence of SEQ ID NO:191. [The present invention 1003] The modified cell of claim 1001 or 1002, wherein the VL comprises the sequence of SEQ ID NO: 192. [The present invention 1004] 1004. The modified cell of any of claims 1001 to 1003, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof. [The present invention 1005] 1005. The modified cell of claim 1004, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. [The present invention 1006] 1001. The modified cell of claim 1001, wherein the transmembrane domain comprises a transmembrane region derived from CD8. [The present invention 1007] 1006. The modified cell of the present invention, wherein the transmembrane region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO:88. [The present invention 1008] The modified cell of claim 1006 or 1007, wherein the transmembrane domain further comprises a hinge region derived from CD8. [The present invention 1009] 1008. The modified cell of the present invention, wherein the hinge region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO:86. [The present invention 1010] The modified cell of any of claims 1006 to 1009, wherein the intracellular domain comprises a 4-1BB signaling domain and a CD3 zeta signaling domain. [The present invention 1011] The modified cell of any of claims 1006 to 1010, wherein the intracellular domain comprises an ICOS signaling domain and a CD3 zeta signaling domain. [The present invention 1012] 10. The modified cell of any of claims 1006 to 1011, wherein the intracellular domain comprises a variant ICOS signaling domain and a CD3 zeta signaling domain. [The present invention 1013] 10. The modified cell of the present invention 1010, wherein the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO:92. [The present invention 1014] 1011. The modified cell of the present invention, wherein the ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID NO:203. [The present invention 1015] 1012. The modified cell of the present invention, wherein the variant ICOS signaling domain comprises the amino acid sequence shown in SEQ ID NO:95. [The present invention 1016] 10. The modified cell of any of claims 10 to 15, wherein the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO:97 or 100. [The present invention 1017] Any of the aforementioned modified cells of the present invention, wherein the dominant negative receptor is a truncated variant of a wild-type protein associated with negative signaling. [The present invention 1018] 1017. The modified cell of the present invention, wherein the truncated variant of the wild-type protein associated with negative signaling comprises the amino acid sequence shown in SEQ ID NO:115. [The present invention 1019] The modified cell of any of claims 1001 to 1018, wherein the switch receptor comprises a first domain derived from a first polypeptide associated with a negative signal; and a second domain derived from a second polypeptide associated with a positive signal. [The present invention 1020] The modified cell of the present invention 1019, wherein the first domain comprises at least a portion of an extracellular domain of a first polypeptide associated with a negative signal, and the second domain comprises at least a portion of an intracellular domain of a second polypeptide associated with a positive signal. [The present invention 1021] Any of the aforementioned modified cells of the present invention, wherein the switch receptor further comprises a transmembrane domain of the switch receptor. [The present invention 1022] The modified cell of the present invention 1021, wherein the transmembrane domain of the switch receptor comprises a transmembrane domain of a first polypeptide associated with a negative signal; or a transmembrane domain of a second polypeptide associated with a positive signal. [The present invention 1023] The modified cell of any of claims 1019 to 1022, wherein the first polypeptide associated with a negative signal is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR. [The present invention 1024] The modified cell of any of claims 1019 to 1023, wherein the second polypeptide associated with a positive signal is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R. [The present invention 1025] The modified cell of any of claims 1019 to 1022, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of CD28; and a second domain comprising at least a portion of the intracellular domain of CD28. [The present invention 1026] 1025. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:117. [The present invention 1027] The modified cell of any of claims 1019 to 1022, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of PD1; and a second domain comprising at least a portion of the intracellular domain of CD28. [The present invention 1028] 1027. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:119. [The present invention 1029] 1027. The modified cell of the invention, wherein the first domain comprises at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132. [The present invention 1030] 1029. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:121. [The present invention 1031] The modified cell of any of claims 1019 to 1022, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of PD1, wherein the extracellular domain comprises an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of CD28. [The present invention 1032] 1031. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:121. [The present invention 1033] 10. The modified cell of any of claims 1019 to 1021, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of PD1, wherein the extracellular domain comprises an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of 4-1BB. [The present invention 1034] The modified cell of the present invention 1033, wherein the switch receptor comprises the amino acid sequence shown in SEQ ID NO:215. [This invention 1035] The modified cell of any of claims 1019 to 1021, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of TIM-3; and a second domain comprising at least a portion of the intracellular domain of CD28. [The present invention 1036] 1035. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:127. [This invention 1037] The modified cell of any of claims 1019 to 1021, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rαl. [The present invention 1038] 1037. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:123. [This invention 1039] The modified cell of any of claims 1019 to 1021, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rβ1. [The present invention 1040] 1039. The modified cell of the present invention, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:125. [This invention 1041] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a dominant-negative receptor comprising the amino acid sequence shown in SEQ ID NO: 115; A modified immune cell or a precursor thereof, comprising: [The present invention 1042] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 213 or 215; A modified immune cell or a precursor thereof, comprising: [This invention 1043] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 117 or 119; A modified immune cell or a precursor thereof, comprising: [This invention 1044] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 121; A modified immune cell or a precursor thereof, comprising: [This invention 1045] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 127; A modified immune cell or a precursor thereof, comprising: [The present invention 1046] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 123; A modified immune cell or a precursor thereof, comprising: [This invention 1047] a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 125; A modified immune cell or a precursor thereof, comprising: [This invention 1048] Any of the modified cells of the present invention which are modified T cells. [This invention 1049] The modified T cells of the present invention 1048, which are autologous cells. [The present invention 1050] The modified T cells of the present invention 1048 are allogeneic cells. [This invention 1051] The modified cell of any of claims 1001 to 1050, which is a cytotoxic T lymphocyte (CTL). [This invention 1052] Any of the modified cells of the present invention that are derived from a human cell. [This invention 1053] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 183; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 184; (b) a second nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor; An isolated nucleic acid comprising: [This invention 1054] 1053. The isolated nucleic acid of the present invention, wherein VH comprises the sequence of SEQ ID NO:191. [This invention 1055] 1053 or 1054, an isolated nucleic acid of the present invention, wherein the VL comprises the sequence of SEQ ID NO:192. [This invention 1056] 1053. The isolated nucleic acid of the present invention, wherein the first nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 246, 248, 250, 252, 254, or 256. [This invention 1057] 1053. The isolated nucleic acid of the present invention, wherein the second nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216. [This invention 1058] 8. The isolated nucleic acid of any one of claims 1053 to 1057, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker. [This invention 1059] 1058. The isolated nucleic acid of claim 1058, wherein the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). [The present invention 1060] 1058. The isolated nucleic acid of claim 1058, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. [This invention 1061] 1060. The isolated nucleic acid of claim 1060, wherein the self-cleaving peptide is a 2A peptide. [This invention 1062] 1061. The isolated nucleic acid of the present invention, wherein the 2A peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), Equine rhinitis A virus 2A (E2A), and Foot-and-Mouth Disease virus 2A (F2A). [This invention 1063] 1061. The isolated nucleic acid of claim 1061, wherein the 2A peptide is T2A. [This invention 1064] 1061. The isolated nucleic acid of claim 1061, wherein the 2A peptide is F2A. [This invention 1065] The isolated nucleic acid of any of claims 1053 to 1064, comprising in the 5' to 3' direction: a first nucleic acid sequence, a linker, and a second nucleic acid sequence. [The present invention 1066] The isolated nucleic acid of any of claims 1053 to 1064, comprising in the 5' to 3' direction: a second nucleic acid sequence, a linker, and a first nucleic acid sequence. [This invention 1067] a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a second nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216; and An isolated nucleic acid comprising: [The present invention 1068] a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a second nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor, comprising the nucleic acid sequence set forth in SEQ ID NO: 116; and An isolated nucleic acid comprising: [This invention 1069] 1068. The isolated nucleic acid of claim 1068, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding T2A. [The present invention 1070] 1068. The isolated nucleic acid of claim 1068, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding F2A. [This invention 1071] An expression construct comprising any one of the isolated nucleic acids of the present inventions 1053 to 1070. [This invention 1072] 1071. An expression construct of the present invention, which is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. [This invention 1073] The expression construct of the present invention 1071 is a lentiviral vector. [This invention 1074] The expression construct of 1073 of the present invention, wherein the lentiviral vector further comprises an EF-la promoter. [This invention 1075] The expression construct of any one of claims 1072 to 1074, wherein the lentiviral vector further comprises a rev response element (RRE). [This invention 1076] The expression construct of any of claims 1072 to 1075, wherein the lentiviral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). [This invention 1077] The expression construct of any one of 1072 to 1076, wherein the lentiviral vector further comprises a cPPT sequence. [This invention 1078] 1077. An expression construct of the present invention, wherein the lentiviral vector further comprises an EF-la promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence. [This invention 1079] The expression construct of any of claims 1072 to 1078, wherein the lentiviral vector is a self-inactivating lentiviral vector. [The present invention 1080] A method for generating a modified immune cell or a precursor thereof according to any of claims 1001 to 1052, comprising introducing into an immune cell one or more of the nucleic acids of any of claims 1053 to 1070 or the expression constructs of any of claims 1071 to 1079. [This invention 1081] A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a modified immune cell of any of claims 1001 to 1052 of the present invention. [This invention 1082] The method of claim 1081, further comprising administering lymphodepleting chemotherapy to the subject. [This invention 1083] The method of claim 1082, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and / or fludarabine. [This invention 1084] 1. A method of treating prostate cancer in a subject in need thereof, the method comprising: administering to the subject lymphodepleting chemotherapy, comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and administering the modified T cells to a subject Including, the modified T cells are a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a dominant-negative receptor comprising the amino acid sequence shown in SEQ ID NO: 115; A method comprising: [This invention 1085] 1. A method of treating metastatic castration-resistant prostate cancer in a subject in need thereof, the method comprising: administering to the subject lymphodepleting chemotherapy, comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and administering the modified T cells to a subject Including, the modified T cells are a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on target cells, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a dominant-negative receptor comprising the amino acid sequence shown in SEQ ID NO: 115; A method comprising: The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings, in which: It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0050] [Figure 1A]Illustrated are results using purified IVT PSMA RNA CAR and full-length PSMA RNA separated on an agarose gel. [Figure 1B] ND444 T cells were electroporated with purified PSMA RNA CAR, and CAR expression was examined by flow cytometry. Mean fluorescence intensity is shown below the graph. [Figure 1C] PSMA expression is shown. Purified full-length PSMA RNA was electroporated into Nalm6 or K562 cells (center and right columns). PSMA expression was examined by flow cytometry. [Figure 1D] The results using mixed PC3.PSMA single cell clones are shown. PC3.PSMA cells (left panel) were subjected to limiting dilution, and seven single colonies were isolated and pooled to form the new cell line, PC3.PSMA.7SC (right panel). PSMA expression was examined by flow cytometry. [Figure 2A] Illustrated are results using various PSMA RNA CARs incubated with tumor cells and subjected to a CD107a assay. Cells were gated by CD3. [Figure 2B] Illustrated are results using various PSMA RNA CARs incubated with tumor cells and subjected to a luciferase-based CTL assay. Results are reported as percent killing based on luciferase activity in wells with tumor alone in the absence of T cells. [Figure 2C] Results are shown using various PSMA RNA CARs incubated with tumor cells and subjected to ELISA assays (IL-2, left column; IFN-γ, right column). [Figure 3A] Illustrated are results using PSMA Lenti CAR constructed and transduced into primary human T cells (MOI=3). CAR expression was examined by flow cytometry on day 8. [Figure 3B]

[0033] Figure 1 shows results using various PSMA LentiCARs incubated with or without tumor cells and subjected to a CD107a assay. Cells were gated by CD3. Results are shown at day 12. [Figure 3C]

[0023] Figure 1 shows the results of using various PSMA LentiCARs incubated with tumor cells and subjected to a luciferase-based CTL assay. Results are reported as percent kill based on luciferase activity in wells containing tumor alone in the absence of T cells. Results are shown at day 12. [Figure 3D] Illustrated are results using various PSMA LentiCARs incubated with PC3 or PC3.PSMA cells and subjected to ELISA assays (IL-2, left column; IFN-γ, right column). Results are shown at day 12. [Figure 4A] Illustrated are results using the switch receptors PD1*PTM.CD28 or PD1.CD28 linked to the respective human PSMA Lenti CAR via F2A and transduced into primary human T cells. PD1 and CAR expression was examined by flow cytometry at day 12. [Figure 4B] Results are illustrated using a dominant-negative (dn) transforming growth factor beta receptor II (TGFRβII) sequence linked via T2A to each human PSMA LentiCAR. dn-TGFRβII-PSMA CAR-transduced T cells were analyzed by flow cytometry on day 7. [Figure 4C] Illustrated are results using various amounts of purified full-length PDL1 RNA electroporated into PC3.PSMA cells and examined for PDL1 expression by flow cytometry on day 13. [Figure 4D] PC3.PSMA cells or PDL1 cells were incubated with electroporated PC3.PSMA cells and subjected to a CD107a assay. Results are shown using various PSMA LentiCARs. Cells were gated by CD3. Results are shown at day 14. [Figure 4E]PC3.PSMA cells or PDL1 cells were incubated with electroporated PC3.PSMA cells and subjected to a CD107a assay. Results are shown using various PSMA LentiCARs. Cells were gated by CD3. Results are shown at day 14. [Figure 4F] PC3.PSMA cells or PDL1 cells were incubated with electroporated PC3.PSMA cells and subjected to a CD107a assay. Results are shown using various PSMA LentiCARs. Cells were gated by CD3. Results are shown at day 14. [Figure 4G] PC3.PSMA cells or PDL1 cells were incubated with electroporated PC3.PSMA cells and subjected to a CD107a assay. Results are shown using various PSMA LentiCARs. Cells were gated by CD3. Results are shown at day 14. [Figure 4H] 1 shows results using various PSMA Lenti CARs incubated with PC3.PSMA cells and subjected to a luciferase-based CTL assay. Results are reported as percent killing based on luciferase activity in wells with tumor alone in the absence of T cells. [Figure 4I] PC3.PSMA cells or PDL1 were incubated with electroporated PC3.PSMA cells and subjected to ELISA assays, showing the results using various PSMA Lenti CARs (IL-2, top panel; IFN-γ, bottom panel). [Figure 5A] Results are shown using the switch receptor PD1.CD28 linked to the respective human PSMA Lenti CAR via F2A transduced into primary human T cells. PD1 and CAR expression was examined by flow cytometry. [Figure 5B] Results are shown using a dominant-negative (dn) TGFRβII sequence linked to the human 2A10 PSMA Lenti CAR via T2A. CAR-transduced T cells were analyzed by flow cytometry. [Figure 5C]Results are shown using various PSMA LentiCARs incubated with PC3.PSMA.7SC cells and subjected to a CD107a assay. Cells were gated by CD3. [Figure 5D] PDL1 were incubated with electroporated PC3.PSMA.7SC cells and subjected to a CD107a assay using various PSMA LentiCARs. Cells were gated by CD3. [Figure 5E]

[0023] Figure 1 shows results using various PSMA LentiCARs incubated with tumor cells and subjected to a luciferase-based CTL assay. Results are reported as percent killing based on luciferase activity in wells with tumor only, in the absence of T cells. [Figure 5F] PC3 cells, PC3.PSMA.7SC cells, or PDL1 cells were incubated with electroporated PC3.PSMA.PDL1 cells, and ELISA assays were performed using various PSMA Lenti CARs (IL-2, top panel; IFN-γ, bottom panel). [Figure 5G] Results using quantitative PCR for PSMA expression are shown. Fold changes (ΔΔCT) were normalized to Nalm6.CBG cells. See Table 1 for abbreviations. [Figure 5H] Results are shown using various PSMA LentiCARs incubated with tumor cells or primary human cells and assayed for CD107a. Cells were gated by CD3. [Figure 5I] Quantitative data from the experiment shown in Figure 5H are shown. HSAEpC: human small airway epithelial cells. HPMEC: human pulmonary microvascular epithelial cells. [Figure 5J] Results are shown using various PSMA LentiCARs incubated with primary human cells and subjected to ELISA assays (IL-2, left column; IFN-γ, right column). HREpC: human renal epithelial cells. HSAEpC: human small airway epithelial cells. HPMEC: human lung microvascular epithelial cells. [Figure 5K]Results are shown using 2x106 PC3.PSMA.7SC cells transduced with click beetles and injected (iv) into mice. 27 days later, 2x106 PSMA CAR-T-positive transduced T cells were injected (iv) into tumor-bearing mice. Bioluminescence imaging (BLI) was performed at multiple time points. The top panel has a minimum mean radiance of 5x105; the bottom panel has a minimum mean radiance of 3x105. [Figure 5L] 5K illustrates the quantitative average radiance. [Figure 6] 1 is a schematic representation of the maps of the dn-TGFRβII PSMA CAR and pTRPE constructs. [Figure 7] Flow cytometry examination of CAR expression in T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS) or co-transduced with 2F5 PSMA CAR together with various switch receptors as indicated is shown. [Figure 8] Flow cytometry examination of PD1 and Tim3 expression in T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS) or co-transduced with 2F5 PSMA CAR together with various switch receptors as indicated is shown. [Figure 9] Graph showing CD107a expression in T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), T cells transduced with PSMA 41BB-CAR alone (41BB), or T cells co-transduced with 2F5 PSMA CAR together with various switch receptors as indicated. UTD means untransduced. [Figure 10] Graph showing Granzyme B expression in T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), T cells transduced with PSMA 41BB-CAR alone (41BB), or T cells co-transduced with 2F5 PSMA CAR together with various switch receptors as indicated. UTD means untransduced. [Figure 11A]IL-2 secretion in T cells transduced with the 2F5 PSMA ICOS-CAR alone (ICOS), the PSMA 41BB-CAR alone (41BB), or co-transduced with the 2F5 PSMA CAR together with various switch receptors as indicated. NTD means non-transduced. [Figure 11B] Graph showing IFN-gamma secretion of T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), T cells transduced with PSMA 41BB-CAR alone (41BB), or T cells co-transduced with 2F5 PSMA CAR together with various switch receptors as indicated. UTD means untransduced. [Figure 12A] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 12B] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 13] Graph showing tumor size in NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14A] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14B] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14C] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14D] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14E] Graph showing quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14F] Graphs showing quantification of bioluminescence (left) and tumor size (right) obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with transduced T cells as indicated. [Figure 14G] Table 1 lists T cells transduced as indicated, ranked from top to bottom, by tumor suppressive ability. ICOSYMNM outperforms WT ICOS. PD1*BB outperforms PD1*CD28 when combined with ICOSz or ICOSzYMNM. [Figure 15A] This graph shows that CART-PSMA-TGFβRdn cells (dnTGFBR2-T2A-Pbbz) demonstrated higher antigen-specific proliferation compared to CART-PSMA(Pbbz) over 42 days of coculture and repeated stimulation with PSMA-expressing tumor cells (arrows). CD19-BBz CART(19bbz) and transduced T cells (mock) were used as controls. [Figure 15B] Graph showing the mean radiance detected in tumor-bearing mice up to 27 days after T cell injection with CART-PSMA-TGFβRdn cells (dnTGFBR2-T2A-Pbbz), CART-PSMA cells (Pbbz), and non-transduced cells (Mock) used as a control. [Figure 15C] Photographs showing tumor regional and total body burden assessed by weekly bioluminescence imaging (BLI). [Figure 16] Describe the clinical trial schema used for Phase 1 clinical trials. [Figure 17]1 shows an assessment of CAR-T cell kinetics by qPCR of CART-PSMA-TGFβRdn DNA in subjects 32816-02, -04, and -05 belong to cohort 1, and subjects 32816-06, -07, and -08 belong to cohort 2. [Figure 18A] 1 is a graph showing significant increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFN gamma) and ferritin associated with a Grade 3 cytokine release syndrome event in subject 32816-06. [Figure 18B] 1 is a graph showing significant increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFN gamma) and ferritin associated with a Grade 3 cytokine release syndrome event in subject 32816-07. [Figure 19] 1 is a graph showing prostate-specific antigen (PSA) response among patients in Cohort 1 and Cohort 2. [Figure 20] Figure 20A is a graph showing PSMA-TGFβRDN CART expression (left y-axis in copies / ug of genomic DNA) and IL-6 levels (right y-axis in pg / ml) in subject 32816-07, demonstrating cytokine release syndrome that developed in the subject one day after infusion. Figure 20B is a graph showing that management of cytokine release syndrome was accompanied by transient PSA decline as measured by C-reactive protein levels (CRP; left y-axis in mg / L) and serum ferritin levels (right y-axis in ng / L). [Figure 21] Graph showing the number of PSMA-positive circulating tumor cells (CTCs) detected in each subject over time. [Figure 22A] FIG. 1 shows two humanized J591 CARs, dnTGF.hJ591VHVK.BBZ and dnTGF.hJ591VKVH.BBZ. [Figure 22B] Flow cytometry examination of CAR and dominant-negative TGFRβII expression in the indicated transduced cells is shown. [Figure 22C]Flow cytometry examination of CD107a expression in the indicated transduced cells is shown. [Figure 23A] 1 is a graph showing the killing efficiency of the indicated transduced cells against co-cultured PC3-PSMA cells. [Figure 23B] 1 is a plot showing the production of various indicated cytokines by various indicated transduced cells co-cultured with PC3-PSMA cells. [Figure 24A] FIG. 1 shows various humanized J591 CARs. [Figure 24B] Shown is the surface expression of the various indicated CARs on transduced T cells as measured using flow cytometry. [Figure 24C] Flow cytometry examination of CD107a expression on the indicated transduced cells when the transduced cells were co-cultured with PC3 or PC3-PSMA cells is shown. [Figure 24D] 1 is a graph showing the killing efficiency of the indicated transduced cells against co-cultured PC3-PSMA cells. [Figure 24E] Graph showing the production of the indicated cytokines by various transduced cells, as indicated, co-cultured with PC3-PSMA cells. [Figure 25] Shown is the surface expression of the various indicated CARs on transduced T cells measured using flow cytometry. [Figure 26A] Flow cytometry examination of dominant-negative TGFRβII and PD1 expression in the indicated transduced cells is shown. [Figure 26B] Flow cytometry examination of CD107a expression in the indicated transduced cells is shown. [Figure 27A] 1 is a graph showing the killing efficiency of the indicated transduced cells against co-cultured PC3-PSMA cells. [Figure 27B] 1 is a graph showing the killing efficiency of the indicated transduced cells against co-cultured PC3 cells. [Figure 28A] 1 is a plot showing IFN-gamma production of various transduced cells as indicated when co-cultured with PC3 or PC3-PSMA cells. [Figure 28B] 1 is a plot showing IL-2 production of various transduced cells, as indicated, co-cultured with PC3 or PC3-PSMA cells. [Figure 29] Photographs depicting tumor site and total body burden in various mice administered the indicated transduced cells, assessed using bioluminescence imaging on the indicated days. [Figure 30A] 1 is a plot showing quantification of the mean flux detected from tumors on the indicated days in various mice receiving the indicated transduced cells. [Figure 30B] 1 is a plot showing quantification of the mean flux detected from tumors at day 21 in various mice receiving the indicated transduced cells. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description The present invention provides compositions and methods for engineered immune cells, such as T cells and NK cells, or their precursor cells, such as engineered T cells, containing chimeric antigen receptors (CARs). In some embodiments, the CAR contains a prostate-specific membrane antigen (PSMA) binding domain (PSMA-CAR) and has affinity for PSMA on target cells, such as prostate cancer cells. In some embodiments, the engineered immune cells contain a PSMA-CAR containing a mouse PSMA-binding domain. In some embodiments, the engineered immune cells contain a PSMA-CAR containing a human PSMA-binding domain. Methods for producing such engineered cells are also provided. In some embodiments, the cells and compositions can be used for adoptive cell therapy, such as adoptive tumor immunotherapy.

[0052] In some embodiments, the provided immune cells contain additional receptors, such as dominant-negative receptors and / or switch receptors, to enhance the efficacy of the immune cells in the tumor microenvironment. Such cells can alter or reduce the effects of immunosuppressive signals in the tumor microenvironment. The modified immune cells of the present invention counteract the upregulation and / or expression of inhibitory receptors or ligands that can negatively regulate T cell activation and function. For example, the expression of certain immune checkpoint proteins, such as PD-1 or PD-L1, on T cells and / or in the tumor microenvironment can reduce the potency and effectiveness of adoptive T cell therapy. For example, the expression of TGF-β on T cells and / or in the tumor microenvironment can reduce the potency and effectiveness of adoptive T cell therapy. Such immunosuppressive signals may otherwise impair certain desirable effector functions associated with adoptive cell therapy. Tumor cells and / or cells in the tumor microenvironment often upregulate immunosuppressive proteins, such as PD-L1, to deliver immunosuppressive signals. Such immunosuppressive proteins may also be upregulated on T cells in the tumor microenvironment, for example, on tumor-infiltrating T cells, which may occur following signal transduction via antigen receptors or certain other activation signals. Such events may contribute to the acquisition of an exhausted phenotype in genetically engineered immune cells (e.g., targeting PSMA) T cells, such as when they are present near other cells expressing such proteins, which may in turn result in reduced functionality. Thus, the modified immune cells of the present invention address the issues of T cell exhaustion and / or lack of T cell persistence that are barriers to the efficacy and therapeutic outcomes of conventional adoptive cell therapy.

[0053] The present invention includes PSMA CAR and its use in cancer treatment.In certain embodiments, the present invention includes human PSMA CAR with dominant negative receptor and / or switch receptor.One of the main obstacles to cancer immunotherapy is the tumor microenvironment.Upregulation of immunosuppressive molecules, such as PD-1, negatively regulates T cell activity.

[0054] The present invention is based on the discovery that T cells comprising a PSMA-CAR and a dominant-negative receptor and / or a switch receptor can evade the effects of immunosuppressive molecules in the tumor microenvironment and provide sustained, potent anti-tumor activity.

[0055] It is understood that the methods described in this disclosure are not limited to the particular methods and / or experimental conditions disclosed herein, as such methods and conditions may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0056] Furthermore, unless otherwise indicated, the experiments described herein use conventional molecular cell biology and immunological techniques within the skill of those skilled in the art.Such techniques are well known to skilled workers and are fully described in literature.For example, see Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, MR Green and J. Sambrook's Molecular Cloning: A Laboratory Manual (Fourth Edition) and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd Edition).

[0057] A. Definition Unless otherwise defined, scientific and technical terms used herein have meanings that are commonly understood by those skilled in the art. In the event of any potential semantic ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Unless otherwise stated, the use of "or" means "and / or." The use of the term "including" and other forms such as "includes" and "included" is non-limiting.

[0058] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and widely used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art, unless otherwise specified, as described in various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are generally performed according to manufacturer's specifications as described herein or as commonly accomplished in the art. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0059] In order that this disclosure may be more readily understood, selected terms are defined below.

[0060] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0061] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are reasonable in practicing the disclosed methods.

[0062] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" particularly refers to T cells undergoing cell division.

[0063] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0064] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or can be an immunoreactive portion of an intact immunoglobulin (e.g., a binding fragment of an antibody). An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0065] The term "antibody fragment" refers to a portion of an intact antibody, and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0066] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0067] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. The α and β light chains refer to the two major antibody light chain isotypes.

[0068] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, the bacteriophage-expressed antibodies described herein. The term should also be taken to mean an antibody produced by synthesis of an antibody-encoding DNA molecule that expresses an antibody protein or an amino acid sequence defining that antibody, where the DNA or amino acid sequence is available and well known in the art, and obtained using DNA or amino acid sequence synthesis techniques.

[0069] As used herein, the term "antigen" or "Ag" is defined as a molecule that provokes an immune response. This immune response may include either or both antibody production or activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule can serve as an antigen, including any protein or peptide.

[0070] Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0071] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into that individual. "Allogeneic" refers to any material derived from a different animal of the same species. "Xenogeneic" refers to any material derived from an animal of a different species.

[0072] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is engineered to be expressed on immune cells and specifically bind to an antigen. CARs can be used as a therapy involving adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific to an antigen or a specific form of the antigen. In some embodiments, CARs have specificity for a selected target, such as cells expressing prostate-specific membrane antigen. CARs can also include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen-binding region.

[0073] "Costimulatory ligand," as that term is used herein, includes a molecule on an antigen-presenting cell (e.g., an artificial APC (aAPC), a dendritic cell, a B cell, etc.) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by engagement of the TCR / CD3 complex with, for example, a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, among others, and ligands that specifically bind to CD83.

[0074] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0075] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0076] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate unless the disease is ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be without the disorder. If left untreated, a disorder does not necessarily cause a further decline in the animal's health.

[0077] The term "downregulation," as used herein, refers to one or more This refers to the reduction or loss of gene expression of several genes.

[0078] "Effective amount" or "therapeutically effective amount," as used interchangeably herein, refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. Immune responses can be readily assessed by numerous art-recognized methods. Those skilled in the art will understand that the amount of a composition administered herein will vary and can be readily determined based on numerous factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the particular compound being administered.

[0079] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0080] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue, or system.

[0081] The term "epitope," as used herein, is defined as a small chemical molecule on an antigen that can elicit an immune response and induce a B-cell response and / or a T-cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. Generally, epitopes are approximately 10 amino acids and / or sugars in size. Preferably, epitopes are about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12 amino acids, and most preferably about 8-10 amino acids. Those skilled in the art will understand that, in general, the overall three-dimensional structure of a molecule, rather than its specific linear sequence, is the primary criterion for antigen specificity, and therefore, this distinguishes one epitope from another. Based on the present disclosure, a peptide of the present invention can be an epitope.

[0082] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0083] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number relative to the number originally present in the culture. In another embodiment, T cells expanded ex vivo are increased in number relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0084] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0085] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating a recombinant polynucleotide.

[0086] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are from human immunoglobulin sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992. "Fully human" refers to an immunoglobulin, such as an antibody, or binding fragment thereof, in which the molecule is entirely of human origin or consists of an amino acid sequence identical to that of the human form of the antibody.

[0087] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. The five members of this protein class are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in bodily secretions, such as saliva, tears, milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in most subjects during the primary immune response. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0088] As used herein, "identity" refers to the identity of the subunit sequence between two amino acid molecules, such as between two polymer molecules, particularly between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10-amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or matched, the two amino acid sequences are 90% identical.

[0089] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0090] The term "immunosuppression" is used herein to refer to the overall reduction of the immune response.

[0091] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0092] " Lentivirus " as used herein refers to a genus of Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, so they are one of the most efficient gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0093] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0094] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0095] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0096] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that a nucleotide sequence encoding a protein, depending on its form, may contain introns.

[0097] The terms "operably linked" or "operatively linked" refer to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0098] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0099] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0100] As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0101] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures.

[0102] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0103] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.), is capable of specifically binding to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0104] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include livestock and pets, such as, for example, ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0105] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0106] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0107] "Graft" refers to a biocompatible lattice or donor tissue, organ, or cells to be transplanted. Examples of grafts include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Grafts can also refer to any material to be administered to a host. For example, grafts can refer to nucleic acids or proteins.

[0108] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0109] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder from which a subject suffers.

[0110] A "vector" is a material composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0111] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0112] B. Chimeric Antigen Receptor The present invention provides compositions and methods for engineered immune cells or their precursor cells, such as engineered T cells, that contain chimeric antigen receptors (CARs).Thus, in some embodiments, immune cells are genetically engineered to express CARs.CARs of the present invention comprise an antigen binding domain, a transmembrane domain, a hinge domain, and an intracellular signaling domain.

[0113] The antigen-binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, both of which are described elsewhere herein, for expression in a cell. In one embodiment, a first nucleic acid sequence encoding the antigen-binding domain is operably linked to a second nucleic acid encoding the transmembrane domain, and is further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0114] The antigen-binding domain described herein can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that can be included in the CAR of the present invention. The CAR of the present invention can also include a spacer domain as described herein. In some embodiments, each of the antigen-binding domain, transmembrane domain, and intracellular domain is separated by a linker.

[0115] antigen-binding domain The antigen binding domain of CAR is the extracellular region of CAR that binds to specific target antigens, including proteins, carbohydrates, and glycolipids.In some embodiments, CAR has affinity for target antigens on target cells.Target antigens can include any kind of protein associated with target cells or its epitope.For example, CAR can have affinity for target antigens on target cells that indicate a specific disease state of target cells.

[0116] In exemplary embodiment, target cell antigen is prostate-specific membrane antigen (PSMA).PSMA is a membrane-bound protein expressed on cell surface, and has been reported to be highly overexpressed in prostate cancer tissue.PSMA expression is directly correlated with tumor grade and stage progression, and is believed to give prostate cancer cells a selective growth advantage.In this way, the exemplary CAR of the present disclosure has affinity for PSMA on target cell.

[0117] As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a mouse target-specific binding domain, e.g., the target-specific binding domain is of mouse origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. In an exemplary embodiment, a CAR of the present disclosure having affinity for PSMA on a target cell may comprise a PSMA-binding domain. In some embodiments, the PSMA-binding domain is a mouse PSMA-binding domain, e.g., the PSMA-binding domain is of mouse origin. In some embodiments, the PSMA-binding domain is a human PSMA-binding domain, e.g., the PSMA-binding domain is of human origin.

[0118] In some embodiments, the CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR may have affinity for one or more target antigens on target cells. In such embodiments, the CAR is a bispecific CAR or a multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR comprising one or more target-specific binding domains with affinity for the same target antigen can also bind to distinct epitopes of the target antigen. When multiple target-specific binding domains are present in a CAR, the binding domains may be arranged in tandem or separated by a linker peptide. For example, in a CAR comprising two target-specific binding domains, the binding domains are covalently linked to each other in a single polypeptide chain via an oligopeptide or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0119] In some embodiments, the antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding fragment (Fab), and a single-chain variable fragment (scFv). In some embodiments, the PSMA-binding domain of the present invention is selected from the group consisting of a PSMA-specific antibody, a PSMA-specific Fab, and a PSMA-specific scFv. In one embodiment, the PSMA-binding domain is a PSMA-specific antibody. In one embodiment, the PSMA-binding domain is a PSMA-specific Fab. In one embodiment, the PSMA-binding domain is a PSMA-specific scFv.

[0120] The antigen-binding domain can comprise any domain that binds to an antigen, and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. In some embodiments, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen-binding domain may depend on the type and number of antigens present on the surface of the target cell.

[0121] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., murine or human) are covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoded linker connecting the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., the PSMA-binding domain) comprises an scFv with an N- to C-terminal configuration of VH-linker-VL. In some embodiments, the antigen-binding domain (e.g., the PSMA-binding domain) comprises an scFv with an N- to C-terminal configuration of VL-linker-VH. Those skilled in the art will be able to select a configuration suitable for use in the present invention.

[0122] The linker is usually rich in glycine for flexibility, and rich in serine or threonine for solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and International Publication No. 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. (GS) n , (GSGGS) n (SEQ ID NO: 1), (GGGS) n (SEQ ID NO: 2), and (GGGGS) n A variety of linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers such as (SEQ ID NO:3), where n represents an integer of at least 1. An exemplary linker sequence is: TIFF2025061674000002.tif18138, etc. A person skilled in the art will be able to select a linker sequence suitable for use in the present invention. In one embodiment, the antigen-binding domain (e.g., PSMA-binding domain) of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are linked to the nucleic acid sequence Amino acid sequence that can be coded for by TIFF2025061674000003.tif11139 They are separated by a linker sequence having TIFF2025061674000004.tif4128.

[0123] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2 (10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0124] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain yields two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0125] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, resulting in two antigen-binding (ab') (bivalent) regions, each of which contains two separate amino acid chains, a portion of a heavy chain for antigen binding and a light (L) chain, linked by an S-linkage, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0126] In some embodiments, the antigen binding domain can be derived from the same species as the species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR can comprise a human antibody or fragment thereof, as described elsewhere herein.

[0127] In an exemplary embodiment, the PSMA-CAR of the invention comprises a PSMA-binding domain, e.g., a PSMA-specific scFv.

[0128] (a) Mouse PSMA-binding domain and its variants In certain embodiments, the PSMA-CAR of the present invention comprises a murine PSMA-binding domain or a variant thereof.

[0129] In certain embodiments, the PSMA-CAR of the present invention comprises the PSMA-binding domain of a non-human PSMA antibody (e.g., a mouse or rat PSMA antibody), or a variant thereof. As is well known in the art, mouse or other non-human antibodies can be produced by immunizing a non-human (e.g., a mouse) with human PSMA or a fragment thereof.

[0130] In one embodiment, the PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000005.tif107144: Mouse J591 PSMA binding domain contained in TIFF2025061674000006.tif31144.

[0131] Acceptable variations of the mouse J591 PSMA-binding domain that maintain PSMA-binding ability will be known to those skilled in the art. For example, in some embodiments, the PSMA-binding domain is a mouse J591 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the mouse J591 PSMA-binding domain contained in SEQ ID NO: 14. In one embodiment, the PSMA-binding domain is a mouse J591 PSMA-binding domain contained in the amino acid sequence set forth in SEQ ID NO: 14.

[0132] In some embodiments, the PSMA-binding domain is a murine J591 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the coding sequence of the murine J591 PSMA-binding domain contained in SEQ ID NO: 15. In one embodiment, the PSMA-binding domain is a murine J591 PSMA-binding domain encoded by a coding sequence contained in the nucleic acid sequence set forth in SEQ ID NO: 15.

[0133] In an exemplary embodiment, the PSMA-CAR of the present invention comprises a PSMA-binding domain, e.g., a PSMA-specific scFv. In one embodiment, the PSMA-binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000007.tif93144: TIFF2025061674000008.tif32144 is the mouse J591 PSMA binding domain.

[0134] Acceptable variations of the mouse J591 PSMA-binding domain that retain binding to human PSMA will be known to those of skill in the art. For example, in some embodiments, the PSMA-binding domain is a mouse J591 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In one embodiment, the PSMA-binding domain is a mouse J591 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0135] In some embodiments, the PSMA-binding domain is a murine J591 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 180. In one embodiment, the PSMA-binding domain is a murine J591 PSMA-binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 180.

[0136] In one embodiment, the mouse J591 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000009.tif45144: Contains the light chain variable region containing TIFF2025061674000010.tif18143.

[0137] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the murine J591 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16. In one embodiment, the murine J591 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:16.

[0138] In some embodiments, the murine J591 PSMA-binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 17. In one embodiment, the murine J591 PSMA-binding domain comprises a light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 17.

[0139] In one embodiment, the murine J591 PSMA binding domain has the amino acid sequence shown below: TIFF2025061674000011.tif18143, containing the light chain variable region described in NCBI GenBank sequence database ID: CCA78125.1.

[0140] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the murine J591 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:181. In one embodiment, the murine J591 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:181. The light chain variable region of the mouse J591 PSMA-binding domain contains three light chain complementarity-determining regions (CDRs). As used herein, "complementarity-determining region" or "CDR" refers to the region of the variable chain of an antigen-binding molecule that binds to a specific antigen. Thus, the mouse J591 PSMA-binding domain may contain a light chain variable region comprising CDR1 represented by the amino acid sequence KASQDVGTAVD (SEQ ID NO: 18); CDR2 represented by the amino acid sequence WASTRHT (SEQ ID NO: 19); and CDR3 represented by the amino acid sequence QQYNSYPLT (SEQ ID NO: 20). Permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a murine J591 PSMA binding domain can comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO:18.For example, a murine J591 PSMA-binding domain can comprise a light chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 19. For example, a murine J591 PSMA-binding domain can comprise a light chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO:20. In one embodiment, the murine J591 PSMA binding domain comprises a light chain variable region comprising three of the CDRs of said light chain variable region.

[0141] In one embodiment, the mouse J591 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below can be encoded by TIFF2025061674000012.tif45144. TIFF2025061674000013.tif18144, including the heavy chain variable region.

[0142] Acceptable variations in the heavy chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the murine J591 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21. In one embodiment, the murine J591 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:21.

[0143] In some embodiments, the murine J591 PSMA-binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 22. In one embodiment, the murine J591 PSMA-binding domain comprises a heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 22.

[0144] In one embodiment, the murine J591 PSMA binding domain has the amino acid sequence shown below: TIFF2025061674000014.tif18144, containing the heavy chain variable region described in NCBI GenBank sequence database ID: CCA78124.1.

[0145] Acceptable variations in the heavy chain variable region while maintaining its contribution to PSMA binding will be known to those of skill in the art. For example, in some embodiments, the murine J591 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:182. In one embodiment, the murine J591 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:182.

[0146] The heavy chain variable region of the mouse J591 PSMA-binding domain contains three heavy chain complementarity-determining regions (CDRs). Thus, the mouse J591 PSMA-binding domain may contain a heavy chain variable region comprising CDR1 represented by the amino acid sequence GYTFTEYTIH (SEQ ID NO: 23); CDR2 represented by the amino acid sequence NINPNNGGTTYNQKFED (SEQ ID NO: 24); and CDR3 represented by the amino acid sequence GWNFDY (SEQ ID NO: 25). Permissible variations in the heavy chain CDRs while maintaining their contribution to human PSMA binding will be known to those skilled in the art. For example, a murine J591 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 23. For example, a murine J591 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 24. For example, a murine J591 PSMA binding domain can comprise a heavy chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO:25.In one embodiment, the murine J591 PSMA binding domain comprises a heavy chain variable region comprising three of the CDRs of said heavy chain variable region.

[0147] In one embodiment, the PSMA-binding domain is a murine J591 PSMA-binding domain comprising an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs:16 and 21.

[0148] In one embodiment, the PSMA-binding domain is a murine J591 PSMA-binding domain comprising an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs:181 and 182.

[0149] (b) Humanized PSMA-binding domain In certain embodiments, the PSMA-CAR of the present invention comprises a humanized variant of the PSMA-binding domain of a non-human PSMA antibody, or a variant or fragment thereof. In certain exemplary embodiments, the PSMA CAR comprises a humanized variant of the mouse J591 antibody that binds to human PSMA. Methods for humanizing mouse antibodies are well known in the art.

[0150] In one embodiment, the PSMA-binding domain is a humanized PSMA-specific binding domain. In a specific embodiment, the PSMA-binding domain is a humanized J591 PSMA-binding domain. In a specific embodiment, the PSMA-binding domain comprises any of the heavy chain variable regions and light chain variable regions disclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1, the disclosures of which are incorporated herein by reference in their entirety. For example, the PSMA-binding domain of the present invention can comprise an scFv comprising any of the heavy chain variable regions and light chain variable regions disclosed herein. Thus, the PSMA-CAR of the present invention comprises a humanized variant of the mouse J591 antibody that binds to human PSMA, as disclosed in WO2017212250A1 and WO2018033749A1.

[0151] In certain embodiments, the PSMA binding domain of the invention can comprise any of the heavy and light chain variable regions shown in Table 19.

[0152] Table 19. Humanized PSMA-binding heavy and light chain variable sequences TIFF2025061674000015.tif79156TIFF2025061674000016.tif231156TIFF2025061674000017.tif166156

[0153] In certain embodiments, the PSMA-CAR of the present disclosure comprises a humanized PSMA-specific binding domain. In one embodiment, the PSMA-binding domain has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000018.tif59144: TIFF2025061674000019.tif31144 is a humanized J591 ("huJ591") binding domain containing

[0154] In certain embodiments, the PSMA binding domain has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000020.tif59144: TIFF2025061674000021.tif31144 is the huJ591 binding domain containing

[0155] In certain embodiments, the PSMA binding domain has the following amino acid sequence: TIFF2025061674000022.tif32144 is the huJ591 binding domain containing

[0156] Acceptable variations of the huJ591 PSMA-binding domain that retain binding to human PSMA will be known to those of skill in the art. For example, in some embodiments, the PSMA-binding domain is a huJ591 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:237, SEQ ID NO:239, or SEQ ID NO:241. In one embodiment, the PSMA-binding domain is a huJ591 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO:237, SEQ ID NO:239, or SEQ ID NO:241.

[0157] In some embodiments, the PSMA-binding domain is a huJ591 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:238 or SEQ ID NO:240. In one aspect, the PSMA-binding domain is a huJ591 PSMA-binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO:238 or SEQ ID NO:240.

[0158] In one embodiment, the huJ591 PSMA binding domain has the following nucleic acid sequence: The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 191, which can be encoded by TIFF2025061674000023.tif45144.

[0159] Acceptable variations of the heavy chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the huJ591 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:191. In one aspect, the huJ591 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:191.

[0160] In some embodiments, the huJ591 PSMA-binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 242. In one aspect, the huJ591 PSMA-binding domain comprises a heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 242.

[0161] The heavy chain variable region of the huJ591 PSMA-binding domain contains three heavy chain complementarity-determining regions (CDRs). Thus, the huJ591 PSMA-binding domain can contain a heavy chain variable region comprising CDR1 represented by the amino acid sequence EYTIH (SEQ ID NO: 243); CDR2 represented by the amino acid sequence NINPNNGGTTYNQKFED (SEQ ID NO: 24); and CDR3 represented by the amino acid sequence GWNFDY (SEQ ID NO: 25). Permissible variations of the heavy chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a huJ591 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 243. For example, a huJ591 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 24. For example, the huJ591 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 25. In one embodiment, the huJ591 PSMA-binding domain comprises a heavy chain variable region comprising the three heavy chain variable region CDRs described above.

[0162] In one embodiment, the huJ591 PSMA binding domain has the following nucleic acid sequence: The antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 192, which can be encoded by TIFF2025061674000024.tif45143.

[0163] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the huJ591 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:192. In one aspect, the huJ591 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:192.

[0164] In some embodiments, the huJ591 PSMA-binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 244. In one aspect, the huJ591 PSMA-binding domain comprises a light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 244.

[0165] The light chain variable region of the huJ591 PSMA-binding domain contains three light chain complementarity-determining regions (CDRs). Thus, the huJ591 PSMA-binding domain can contain a light chain variable region containing CDR1 represented by the amino acid sequence KASQDVGTAVD (SEQ ID NO: 18); CDR2 represented by the amino acid sequence WASTRHT (SEQ ID NO: 19); and CDR3 represented by the amino acid sequence QQYNSYPLT (SEQ ID NO: 20). Permissible variations of the light chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a huJ591 PSMA-binding domain can comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 18. For example, a huJ591 PSMA-binding domain can comprise a light chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 19. For example, the huJ591 PSMA-binding domain can comprise a light chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 20. In one embodiment, the huJ591 PSMA-binding domain comprises a light chain variable region comprising the three light chain variable region CDRs described above.

[0166] (c) Human PSMA-binding domain In certain embodiments, the PSMA-CAR of the present invention comprises a PSMA-binding domain of a human PSMA antibody or a variant thereof. In one embodiment, the PSMA-binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000025.tif107144: TIFF2025061674000026.tif38142 is a human 1C3 PSMA binding domain containing

[0167] Acceptable variations of the human 1C3 PSMA-binding domain that maintain binding to human PSMA will be known to those skilled in the art. For example, in some embodiments, the PSMA-binding domain is a human 1C3 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In one embodiment, the PSMA-binding domain is a human 1C3 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO:26.

[0168] In some embodiments, the PSMA-binding domain is a human 1C3 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 27. In one embodiment, the PSMA-binding domain is a human 1C3 PSMA-binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 27.

[0169] In one embodiment, the human 1C3 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000027.tif52144: TIFF2025061674000028.tif18144, including the heavy chain variable region.

[0170] Acceptable variations in the heavy chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 1C3 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28. In one embodiment, the human 1C3 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28.

[0171] In some embodiments, the human 1C3 PSMA-binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 29. In one embodiment, the human 1C3 PSMA-binding domain comprises a heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 29.

[0172] The heavy chain variable region of the human 1C3 PSMA binding domain comprises three heavy chain complementarity determining regions (CDRs). Thus, the human 1C3 PSMA binding domain may comprise a heavy chain variable region comprising CDR1 represented by the amino acid sequence SYAMH (SEQ ID NO: 30); CDR2 represented by the amino acid sequence VISYDGNNKYYADSVKG (SEQ ID NO: 31); and CDR3 represented by the amino acid sequence AVPWGSRYYYYGMDV (SEQ ID NO: 32). Permissible variations in the heavy chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 1C3 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 30. For example, a human 1C3 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 31. For example, a human 1C3 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 32. In one embodiment, the human 1C3 PSMA-binding domain comprises a heavy chain variable region comprising three of the CDRs of said heavy chain variable region.

[0173] In one embodiment, the human 1C3 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000029.tif45144: Contains the light chain variable region containing TIFF2025061674000030.tif18144.

[0174] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 1C3 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:33. In one embodiment, the human 1C3 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:33.

[0175] In some embodiments, the human 1C3 PSMA-binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 34. In one embodiment, the human 1C3 PSMA-binding domain comprises a light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 34.

[0176] The light chain variable region of the human 1C3 PSMA-binding domain comprises three light chain complementarity-determining regions (CDRs). Thus, the human 1C3 PSMA-binding domain may comprise a light chain variable region comprising CDR1 represented by the amino acid sequence RASQGISSALA (SEQ ID NO: 35); CDR2 represented by the amino acid sequence DASSLES (SEQ ID NO: 36); and CDR3 represented by the amino acid sequence QQFNSYPLT (SEQ ID NO: 37). Permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 1C3 PSMA-binding domain can comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 35. For example, a human 1C3 PSMA-binding domain can comprise a light chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 36. For example, a human 1C3 PSMA-binding domain can comprise a light chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 37. In one embodiment, the human 1C3 PSMA-binding domain comprises a light chain variable region comprising three of the CDRs of said light chain variable region.

[0177] In one embodiment, the PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000031.tif107144: TIFF2025061674000032.tif31143 is the human 2A10 PSMA binding domain.

[0178] Acceptable variations of the human 2A10 PSMA-binding domain that maintain binding to human PSMA will be known to those skilled in the art. For example, in some embodiments, the PSMA-binding domain is a human 2A10 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:38. In one embodiment, the PSMA-binding domain is a human 2A10 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO:38.

[0179] In some embodiments, the PSMA-binding domain is a human 2A10 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 39. In one embodiment, the PSMA-binding domain is a human 2A10 PSMA-binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 39.

[0180] In one embodiment, the human 2A10 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000033.tif52144: TIFF2025061674000034.tif18144, including the heavy chain variable region.

[0181] Acceptable variations in the heavy chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 2A10 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:40. In one embodiment, the human 2A10 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:40.

[0182] In some embodiments, the human 2A10 PSMA-binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 41. In one embodiment, the human 2A10 PSMA-binding domain comprises a heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 41.

[0183] The heavy chain variable region of the human 2A10 PSMA-binding domain contains three heavy chain complementarity-determining regions (CDRs). Thus, the human 2A10 PSMA-binding domain may contain a heavy chain variable region comprising CDR1 represented by the amino acid sequence SNWIG (SEQ ID NO: 42); CDR2 represented by the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 43); and CDR3 represented by the amino acid sequence QTGFLWSSDL (SEQ ID NO: 44). Permissible variations in the heavy chain CDRs while maintaining their contribution to human PSMA binding will be known to those skilled in the art. For example, a human 2A10 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 42. For example, a human 2A10 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 43. For example, a human 2A10 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 44. In one embodiment, the human 2A10 PSMA-binding domain comprises a heavy chain variable region comprising three of the CDRs of said heavy chain variable region.

[0184] In one embodiment, the human 2A10 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000035.tif45144: Contains the light chain variable region containing TIFF2025061674000036.tif18142.

[0185] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 2A10 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:45. In one embodiment, the human 2A10 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:45.

[0186] In some embodiments, the human 2A10 PSMA-binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 46. In one embodiment, the human 2A10 PSMA-binding domain comprises a light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 46.

[0187] The light chain variable region of the human 2A10 PSMA-binding domain contains three light chain complementarity-determining regions (CDRs). Thus, the human 2A10 PSMA-binding domain may contain a light chain variable region comprising CDR1 represented by the amino acid sequence CRASQDISSAL (SEQ ID NO: 47); CDR2 represented by the amino acid sequence YDASSLES (SEQ ID NO: 48); and CDR3 represented by the amino acid sequence CQQFNSYPLT (SEQ ID NO: 49). Permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 2A10 PSMA-binding domain can comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 47. For example, a human 2A10 PSMA-binding domain can comprise a light chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 48. For example, a human 2A10 PSMA-binding domain can comprise a light chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 49. In one embodiment, the human 2A10 PSMA-binding domain comprises a light chain variable region comprising three of the CDRs of said light chain variable region.

[0188] In one embodiment, the PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000037.tif106144: TIFF2025061674000038.tif38143 is the human 2F5 PSMA binding domain.

[0189] Acceptable variations of the human 2F5 PSMA-binding domain that maintain binding to human PSMA will be known to those skilled in the art. For example, in some embodiments, the PSMA-binding domain is a human 2F5 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:50. In one embodiment, the PSMA-binding domain is a human 2F5 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO:50.

[0190] In some embodiments, the PSMA-binding domain is a human 2F5 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 51. In one embodiment, the PSMA-binding domain is a human 2F5 PSMA-binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 51.

[0191] In one embodiment, the human 2F5 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000039.tif52144: TIFF2025061674000040.tif18142 containing the heavy chain variable region.

[0192] Acceptable variations in the heavy chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 2F5 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:52. In one embodiment, the human 2F5 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:52.

[0193] In some embodiments, the human 2F5 PSMA-binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 53. In one embodiment, the human 2F5 PSMA-binding domain comprises a heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 53.

[0194] The heavy chain variable region of the human 2F5 PSMA-binding domain contains three heavy chain complementarity-determining regions (CDRs). Thus, the human 2F5 PSMA-binding domain may contain a heavy chain variable region containing CDR1 represented by the amino acid sequence SNWIG (SEQ ID NO: 54); CDR2 represented by the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 55); and CDR3 represented by the amino acid sequence QTGFLWSFDL (SEQ ID NO: 56). Permissible variations in the heavy chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 2F5 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 54. For example, a human 2F5 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 55. For example, a human 2F5 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 56. In one embodiment, the human 2F5 PSMA-binding domain comprises a heavy chain variable region comprising three of the CDRs of said heavy chain variable region.

[0195] In one embodiment, the human 2F5 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000041.tif45144: Contains the light chain variable region containing TIFF2025061674000042.tif18144.

[0196] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 2F5 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:57. In one embodiment, the human 2F5 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:57.

[0197] In some embodiments, the human 2F5 PSMA-binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 58. In one embodiment, the human 2F5 PSMA-binding domain comprises a light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 58.

[0198] The light chain variable region of the human 2F5 PSMA-binding domain comprises three light chain complementarity-determining regions (CDRs). Thus, the human 2F5 PSMA-binding domain may comprise a light chain variable region comprising CDR1 represented by the amino acid sequence RASQDISSALA (SEQ ID NO: 59); CDR2 represented by the amino acid sequence DASSLES (SEQ ID NO: 60); and CDR3 represented by the amino acid sequence QQFNSYPLT (SEQ ID NO: 61). Permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 2F5 PSMA-binding domain can comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 59. For example, a human 2F5 PSMA-binding domain can comprise a light chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 60. For example, a human 2F5 PSMA-binding domain can comprise a light chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 61. In one embodiment, the human 2F5 PSMA-binding domain comprises a light chain variable region comprising three of the CDRs of said light chain variable region.

[0199] In one embodiment, the PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000043.tif107144: TIFF2025061674000044.tif38143 is a human 2C6 PSMA binding domain containing

[0200] Acceptable variations of the human 2C6 PSMA-binding domain that maintain binding to human PSMA will be known to those skilled in the art. For example, in some embodiments, the PSMA-binding domain is a human 2C6 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:62. In one embodiment, the PSMA-binding domain is a human 2C6 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO:62.

[0201] In some embodiments, the PSMA-binding domain is a human 2C6 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 63. In one embodiment, the PSMA-binding domain is a human 2C6 PSMA-binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 63.

[0202] In one embodiment, the human 2C6 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000045.tif52144: TIFF2025061674000046.tif18143, including the heavy chain variable region.

[0203] Acceptable variations in the heavy chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 2C6 PSMA-binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:64. In one embodiment, the human 2C6 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:64.

[0204] In some embodiments, the human 2C6 PSMA-binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 65. In one embodiment, the human 2C6 PSMA-binding domain comprises a heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 65.

[0205] The heavy chain variable region of the human 2C6 PSMA-binding domain contains three heavy chain complementarity-determining regions (CDRs). Thus, the human 2C6 PSMA-binding domain may contain a heavy chain variable region comprising CDR1 represented by the amino acid sequence TNYWI (SEQ ID NO: 66); CDR2 represented by the amino acid sequence GIIYPGDSDTRYSPSFQG (SEQ ID NO: 67); and CDR3 represented by the amino acid sequence SPGYTSSWTS (SEQ ID NO: 68). Permissible variations in the heavy chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 2C6 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 66. For example, a human 2C6 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 67. For example, a human 2C6 PSMA-binding domain can comprise a heavy chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 68. In one embodiment, the human 2C6 PSMA-binding domain comprises a heavy chain variable region comprising three of the CDRs of said heavy chain variable region.

[0206] In one embodiment, the human 2C6 PSMA binding domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000047.tif45144: Contains the light chain variable region containing TIFF2025061674000048.tif18144.

[0207] Acceptable variations in the light chain variable region while maintaining its contribution to human PSMA binding will be known to those of skill in the art. For example, in some embodiments, the human 2C6 PSMA-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:69. In one embodiment, the human 2C6 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:69.

[0208] In some embodiments, the human 2C6 PSMA-binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 70. In one embodiment, the human 2C6 PSMA-binding domain comprises a light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 70.

[0209] The light chain variable region of the human 2C6 PSMA-binding domain contains three light chain complementarity-determining regions (CDRs). Thus, the human 2C6 PSMA-binding domain may contain a light chain variable region comprising CDR1 represented by the amino acid sequence CRASQSVSSYL (SEQ ID NO: 71); CDR2 represented by the amino acid sequence YDASNRAT (SEQ ID NO: 72); and CDR3 represented by the amino acid sequence CQQRSNWPLFT (SEQ ID NO: 73). Permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding will be known to those skilled in the art. For example, a human 2C6 PSMA-binding domain can comprise a light chain variable region comprising a CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 71. For example, a human 2C6 PSMA-binding domain can comprise a light chain variable region comprising a CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 72. For example, a human 2C6 PSMA-binding domain can comprise a light chain variable region comprising a CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 73. In one embodiment, the human 2C6 PSMA-binding domain comprises a light chain variable region comprising three of the CDRs of said light chain variable region.

[0210] Transmembrane domain The CAR of the present invention (e.g., PSMA-CAR) can comprise a transmembrane domain that connects the antigen-binding domain of the CAR with the intracellular domain of the CAR. The transmembrane domain of the subject CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor cell). The transmembrane domain is for insertion into a cell membrane, for example, a eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0211] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains with the transmembrane domains of the same or different surface membrane proteins, minimizing interaction with other members of the receptor complex.

[0212] Transmembrane domain can be derived from either natural or synthetic source.When source is natural, domain can be derived from any membrane-binding protein or transmembrane protein, for example, type I transmembrane protein.When source is synthetic, transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane domains of particular use in the present invention include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane regions of) the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triad will be found at each end of the synthetic transmembrane domain.

[0213] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that can be included in a subject CAR.

[0214] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise a hinge region. The hinge region of a CAR is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component of a CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that can be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0215] In some embodiments, the CAR of the present disclosure comprises a hinge region that connects the antigen-binding domain to the transmembrane domain, which in turn connects it to the intracellular domain. The hinge region preferably can assist the antigen-binding domain in recognizing and binding to the target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, allowing the antigen-binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows the hinge region to adopt many different conformations.

[0216] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0217] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

[0218] A suitable hinge region can be readily selected and can be any of several suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0219] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 1) and (GGGS) n (SEQ ID NO:2), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively conformationally undefined and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the φ-ψ space than alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions include, but are not limited to: It can contain amino acid sequences such as TIFF2025061674000049.tif18139.

[0220] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions are known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, an immunoglobulin hinge region may have the following amino acid sequence: It can contain one of the following: TIFF2025061674000050.tif52138.

[0221] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be substituted with Tyr, thereby causing the hinge region to comprise the sequence EPKSCDKTYTCPPCP (SEQ ID NO:85); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0222] The transmembrane domain may be combined with any hinge region and / or may comprise one or more transmembrane domains described herein. In one embodiment, the transmembrane domain comprises a CD8 transmembrane domain. In one embodiment, the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane domain. In some embodiments, the subject CAR comprises: The nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000051.tif18143: Contains the CD8 hinge region with TIFF2025061674000052.tif4143.

[0223] Acceptable variations in transmembrane domains while retaining their intended function will be known to those of skill in the art. For example, in some embodiments, a CAR of the present invention comprises a transmembrane domain comprising a CD8 hinge region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:86. In one embodiment, a CAR comprises a transmembrane domain comprising a CD8 hinge region comprising the amino acid sequence set forth in SEQ ID NO:86.

[0224] In some embodiments, a CAR of the present invention comprises a transmembrane domain comprising a CD8 hinge region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 87. In one embodiment, a CAR comprises a transmembrane domain comprising a CD8 hinge region encoded by the nucleic acid sequence set forth in SEQ ID NO: 87.

[0225] In some embodiments, the subject CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000053.tif11142: Contains the CD8 transmembrane domain with TIFF2025061674000054.tif4128.

[0226] Acceptable variations in transmembrane domains while retaining their intended function will be known to those of skill in the art. For example, in some embodiments, a CAR of the present invention comprises a transmembrane domain comprising a CD8 transmembrane domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:88. In one embodiment, a CAR comprises a transmembrane domain comprising a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:88.

[0227] In some embodiments, a CAR of the present invention comprises a transmembrane domain comprising a CD8 transmembrane domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 89. In one embodiment, a CAR comprises a transmembrane domain comprising a CD8 transmembrane domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 89.

[0228] In some embodiments, the transmembrane domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000055.tif31144: Contains the CD8 hinge region and CD8 transmembrane domain with TIFF2025061674000056.tif11142.

[0229] Acceptable variations in transmembrane domains while retaining their intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:90. In one embodiment, a CAR comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:90.

[0230] In some embodiments, a CAR of the present invention comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 91. In one embodiment, a CAR comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 91.

[0231] A spacer domain may be incorporated between the extracellular and transmembrane domains of a CAR, or between the intracellular and transmembrane domains of a CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to either the extracellular or intracellular domain within a polypeptide chain. A spacer domain may contain up to 300 amino acids, e.g., 10-100 amino acids, or 25-50 amino acids. In some embodiments, a spacer domain may be a short oligopeptide or polypeptide linker, e.g., 2-10 amino acids in length. For example, a glycine-serine dyad provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of a CAR of interest.

[0232] Intracellular signaling domains The CAR of the present invention also comprises an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of CAR is responsible for activating at least one effector function of the cell (e.g., immune cell) in which CAR is expressed. The intracellular signaling domain transmits effector function signals and directs the cell (e.g., immune cell) to carry out its special function, such as damaging and / or destroying target cells.

[0233] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecules that act in concert to initiate signaling in T cells, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0234] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5, and CD28. In one aspect, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic receptor, and combinations thereof.

[0235] In one embodiment, the intracellular signaling domain of the CAR comprises at least one signaling domain from any portion of one or more costimulatory molecules, for example, CD3, CD8, CD27, CD28, ICOS, 4-IBB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0236] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RiIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD lib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0237] Additional examples of intracellular domains include the intracellular signaling domains of several different immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0238] Intracellular signaling domains suitable for use in the subject CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cellular behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerizing agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently linked to the membrane-bound CAR, but instead is diffused in the cytoplasm.

[0239] Intracellular signaling domains suitable for use in the subject CARs of the present invention comprise immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject CAR comprises three ITAM motifs.

[0240] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0241] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain does not need to contain the entire sequence of the protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0242] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer-activating receptor-associated protein; killer-activating receptor-associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high-affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high-affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (B cell antigen receptor complex-associated protein alpha chain; also known as CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a DAP10 / CD28-type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0243] Typically, the entire intracellular signaling domain can be used, although in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain so long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0244] The intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0245] In one embodiment, the intracellular domain of a subject CAR has the nucleic acid sequence shown below: TIFF2025061674000057.tif18143 or the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000058.tif18143: Contains 4-1BB domain containing TIFF2025061674000059.tif4137.

[0246] Acceptable variations of the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:92. In one embodiment, a CAR comprises an intracellular domain comprising a 4-1BB domain comprising the amino acid sequence set forth in SEQ ID NO:92.

[0247] In some embodiments, a CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 93 or 94. In one embodiment, a CAR comprises an intracellular domain comprising a 4-1BB domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 93 or 94.

[0248] In one embodiment, the intracellular domain of a subject CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000060.tif18143: Contains ICOS domain including TIFF2025061674000061.tif4128.

[0249] Acceptable variations of the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising an ICOS domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:203. In one embodiment, a CAR comprises an intracellular domain comprising an ICOS domain comprising the amino acid sequence set forth in SEQ ID NO:203.

[0250] In some embodiments, a CAR of the present invention comprises an intracellular domain comprising an ICOS domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 204. In one embodiment, a CAR comprises an intracellular domain comprising an ICOS domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 204.

[0251] In one embodiment, the intracellular domain of a subject CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000062.tif18144: TIFF2025061674000063.tif4128 containing a variant ICOS domain.

[0252] The variant ICOS domain is also referred to herein as ICOS(YMNM).

[0253] Acceptable variations of the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising an ICOS domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:95. In one embodiment, a CAR comprises an intracellular domain comprising an ICOS domain comprising the amino acid sequence set forth in SEQ ID NO:95.

[0254] In some embodiments, a CAR of the present invention comprises an intracellular domain comprising an ICOS domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 96. In one embodiment, a CAR comprises an intracellular domain comprising an ICOS domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 96.

[0255] In one embodiment, the intracellular domain of a subject CAR has the nucleic acid sequence shown below: TIFF2025061674000064.tif45143 or the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000065.tif45143: Contains CD3 zeta domain containing TIFF2025061674000066.tif18144.

[0256] Acceptable variations in the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:97. In one embodiment, a CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO:97.

[0257] In some embodiments, a subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 98 or 99. In one embodiment, a subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 98 or 99.

[0258] The CD3 zeta domain has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000067.tif45144: TIFF2025061674000068.tif18144.

[0259] Acceptable variations in the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 100. In one embodiment, a CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0260] In some embodiments, a subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 101. In one embodiment, a subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 101.

[0261] In one embodiment, the CAR comprises an intracellular domain comprising a CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO:97 or 100.

[0262] In one exemplary embodiment, the intracellular domain of the subject CAR has the nucleic acid sequence shown below: TIFF2025061674000069.tif65144 or the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000070.tif66144: TIFF2025061674000071.tif25143 containing the 4-1BB domain and the CD3 zeta domain.

[0263] Acceptable variations in the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102. In one embodiment, a CAR comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, comprising the amino acid sequence set forth in SEQ ID NO: 102.

[0264] In some embodiments, a CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 103 or 104. In one embodiment, a CAR comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain encoded by a nucleic acid sequence set forth in SEQ ID NO: 103 or 104.

[0265] In one exemplary embodiment, the intracellular domain of the subject CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000072.tif59143: TIFF2025061674000073.tif25144 containing ICOS domain and CD3 zeta domain.

[0266] Acceptable variations in the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, the intracellular domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 205. In one embodiment, a CAR comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, the intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 205.

[0267] In some embodiments, a CAR of the present invention comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 206. In one embodiment, a CAR comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 206.

[0268] In one exemplary embodiment, the intracellular domain of the subject CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000074.tif59144: TIFF2025061674000075.tif25143 containing variant ICOS domain and CD3 zeta domain.

[0269] Acceptable variations in the intracellular domain while maintaining its intended function will be known to those skilled in the art. For example, in some embodiments, a CAR of the present invention comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain, comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 207. In one embodiment, a CAR comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain, comprising the amino acid sequence set forth in SEQ ID NO: 207.

[0270] In some embodiments, a CAR of the present invention comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 208. In one embodiment, a CAR comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 208.

[0271] CAR sequence The CAR of interest of the present invention may be selected from the group consisting of J591 murine PSMA-CAR, humanized J591 PSMA-CAR, 1C3 human PSMA-CAR, 2A10 human PSMA-CAR, 2F5 human PSMA-CAR, and 2C6 human PSMA-CAR.

[0272] In one embodiment, the CAR of interest of the present invention is a J591 mouse PSMA-CAR. In one embodiment, the J591 mouse PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000076.tif196144: Includes TIFF2025061674000077.tif66144.

[0273] In one embodiment, the CAR of the present invention is a humanized PSMA-CAR, e.g., a humanized J591 PSMA-CAR. In such an embodiment, the humanized PSMA-CAR comprises any of the heavy chain variable regions and light chain variable regions disclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1. For example, the humanized PSMA-CAR of the present invention can comprise an scFv comprising any of the heavy chain variable regions and light chain variable regions disclosed herein, see, for example, the sequences shown in Table 19 of the present disclosure.

[0274] In one embodiment, a subject CAR of the present invention is a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000078.tif120144TIFF2025061674000079.tif66144: Includes TIFF2025061674000080.tif66144.

[0275] In one embodiment, a subject CAR of the present invention is a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000081.tif65144TIFF2025061674000082.tif121144: Includes TIFF2025061674000083.tif66144.

[0276] In one embodiment, a subject CAR of the present invention is a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000084.tif203144: Includes TIFF2025061674000085.tif66144.

[0277] In one embodiment, a subject CAR of the present invention is a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000086.tif204144: Includes TIFF2025061674000087.tif66144.

[0278] In one embodiment, a subject CAR of the present invention is a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000088.tif106144TIFF2025061674000089.tif80144: Includes TIFF2025061674000090.tif66144.

[0279] In one embodiment, a subject CAR of the present invention is a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR has the following nucleic acid sequence: The following amino acid sequence can be encoded by TIFF2025061674000091.tif51144TIFF2025061674000092.tif134144: Includes TIFF2025061674000093.tif66144.

[0280] In one embodiment, the CAR of interest of the present invention is a 1C3 human PSMA-CAR. In one embodiment, the 1C3 human PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000094.tif196144: Includes TIFF2025061674000095.tif66144.

[0281] In one embodiment, the CAR of interest of the present invention is a 2A10 human PSMA-CAR. In one embodiment, the 2A10 human PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000096.tif196144: Includes TIFF2025061674000097.tif66143.

[0282] In one embodiment, the CAR of interest of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000098.tif196144: TIFF2025061674000099.tif66144 containing the 4-1BB domain and the CD3 zeta domain.

[0283] In one embodiment, the CAR of interest of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000100.tif190144: TIFF2025061674000101.tif66144 containing ICOS domain and CD3 zeta domain.

[0284] In one embodiment, the CAR of interest of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000102.tif190144: TIFF2025061674000103.tif66143 containing variant ICOS domain and CD3 zeta domain.

[0285] In one embodiment, the CAR of interest of the present invention is a 2C6 human PSMA-CAR. In one embodiment, the 2C6 human PSMA-CAR has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000104.tif196144: Includes TIFF2025061674000105.tif66144.

[0286] Permissible variations in the sequence of a subject CAR while retaining its function will be known to those of skill in the art.

[0287] For example, in some embodiments, a CAR of the present invention is a J591 murine PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 105. In one embodiment, the CAR is a J591 murine PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 105.

[0288] For example, in some embodiments, a CAR of interest of the present invention is a humanized J591 PSMA-CAR. The humanized J591 PSMA-CAR comprises a humanized J591 PSMA-binding domain comprising heavy and light chain variable regions selected from any of the heavy and light chain variable region sequences shown in Table 19. In some embodiments, the humanized J591 PSMA-CAR comprises a 4-1BB domain and a CD3 zeta domain.

[0289] For example, in some embodiments, a subject CAR of the invention is a huJ591 PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 245, 247, 249, 251, 253, or 255. In one aspect, the CAR is a huJ591 PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 245, 247, 249, 251, 253, or 255.

[0290] For example, in some embodiments, a CAR of the present invention is a 1C3 human PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107. In one embodiment, the CAR is a 1C3 human PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 107.

[0291] For example, in some embodiments, a CAR of interest of the present invention is a 2A10 human PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 109. In one embodiment, the CAR is a 2A10 human PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 109.

[0292] For example, in some embodiments, the CAR of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain that comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain that comprises the amino acid sequence set forth in SEQ ID NO: 111. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain that comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 209. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain that comprise the amino acid sequence set forth in SEQ ID NO: 209.In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain that comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 211. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain that comprise the amino acid sequence set forth in SEQ ID NO: 211. For example, in some embodiments, a CAR of the present invention is a 2C6 human PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 113. In one embodiment, the CAR is a 2C6 human PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 113.

[0293] In some embodiments, the CAR of interest is a J591 mouse PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 106. In one embodiment, the CAR is a J591 mouse PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 106.

[0294] For example, in some embodiments, a subject CAR of the invention is a huJ591 PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 246, 248, 250, 252, 254, or 256. In one aspect, the CAR is a huJ591 PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 246, 248, 250, 252, 254, or 256.

[0295] For example, in some embodiments, a CAR of interest of the present invention is a 1C3 human PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 108. In one embodiment, the CAR is a 1C3 human PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 108. For example, in some embodiments, a CAR of the present invention is a 2A10 human PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 110. In one embodiment, the CAR is a 2A10 human PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 110. For example, in some embodiments, a CAR of the present invention is a 2F5 human PSMA-CAR.In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 112. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 112. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 210. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 210.In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 212. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 212. For example, in some embodiments, a CAR of interest is a 2C6 human PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 114. In one embodiment, the CAR is a 2C6 human PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 114.

[0296] In certain embodiments, the CAR of the present invention may comprise any one of the amino acid sequences corresponding to SEQ ID NOs: 209, 211, 227-236, 245, 247, 249, 251, 253, or 255.

[0297] Table 20: Exemplary CAR sequences TIFF2025061674000106.tif111148TIFF2025061674000107.tif230148TIFF2025061674000108.tif230148TIFF2025061674000109.tif23014 8TIFF2025061674000110.tif230148TIFF2025061674000111.tif230148TIFF2025061674000112.tif230148TIFF2025061674000113.tif88148

[0298] Thus, the present invention provides engineered immune cells or their precursor cells, e.g., engineered T cells, comprising a chimeric antigen receptor (CAR) that has affinity for prostate-specific membrane antigen (PSMA) on target cells (e.g., prostate cancer cells). In some embodiments, the CAR comprises a PSMA-binding domain. In some embodiments, the CAR comprises a mouse PSMA-binding domain. In one embodiment, the CAR comprises a J591 mouse PSMA-binding domain. In one embodiment, the CAR comprises a humanized J591 PSMA-binding domain. In some embodiments, the CAR comprises a human PSMA-binding domain selected from the group consisting of 1C3, 2A10, 2F5, and 2C6 human PSMA-binding domains.

[0299] Therefore, the CAR of the present invention comprises a PSMA binding domain and a transmembrane domain. In one embodiment, the CAR comprises a PSMA binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 hinge region. In one embodiment, the CAR comprises a PSMA binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 transmembrane domain. In one embodiment, the CAR comprises a PSMA binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane domain.

[0300] Thus, the CAR of the present invention comprises a PSMA binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, the CAR comprises a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a 4-1BB domain. In one embodiment, the CAR comprises a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a CD3 zeta domain. In one embodiment, the CAR comprises a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a 4-1BB domain and a CD3 zeta domain.

[0301] C. Dominant-Negative Receptors and Switch Receptors The present invention provides compositions and methods for engineered immune cells or their precursor cells, e.g., engineered T cells, comprising dominant-negative receptors and / or switch receptors. Accordingly, in some embodiments, immune cells are genetically engineered to express dominant-negative receptors and / or switch receptors. As used herein, the term "dominant-negative receptor" refers to a molecule designed to reduce the effect of a negative signaling molecule, e.g., on engineered immune cells of the present invention. Dominant-negative receptors of the present invention may bind to negative signaling molecules, e.g., TGF-β or PD-1, via their extracellular domains associated with negative signals, thereby reducing the effects of the negative signaling molecules. Such dominant-negative receptors are described herein. For example, engineered immune cells comprising dominant-negative receptors may bind to negative signaling molecules in the microenvironment of the engineered immune cells, thereby reducing the effects that the negative signaling molecules may have on the engineered immune cells.

[0302] In addition to reducing the effect of negative signaling molecules, the switch receptors of the present invention may be designed to convert negative signals into positive signals by including an intracellular domain associated with a positive signal. Switch receptors designed to convert negative signals into positive signals are described herein. Thus, the switch receptors include an extracellular domain associated with a negative signal and / or an intracellular domain associated with a positive signal.

[0303] Tumor cells generate an immunosuppressive microenvironment that serves to protect them from immune recognition and elimination. This immunosuppressive microenvironment can limit the effectiveness of immunosuppressive therapies, such as CAR-T cell therapy. The secreted cytokine transforming growth factor-β (TGFβ) directly inhibits the function of cytotoxic T cells and additionally induces the formation of regulatory T cells, further suppressing the immune response. T cell immunosuppression by TGFβ in the context of prostate cancer has previously been demonstrated (Donkor et al., 2011; Shalapour et al., 2015). To reduce the immunosuppressive effects of TGFβ, immune cells can be modified to express a dominant-negative receptor for TGF-β.

[0304] In some embodiments, the dominant-negative receptor is a truncated variant of a wild-type protein associated with negative signaling. In some embodiments, the dominant-negative receptor is a dominant-negative receptor for TGF-β. Thus, in some embodiments, the dominant-negative receptor for TGF-β is a truncated variant of a wild-type TGF-β receptor. In some embodiments, the dominant-negative receptor is a truncated dominant-negative variant of TGF-β receptor type II (TGFβRII-DN). In one embodiment, TGFβRII-DN has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000114.tif80144: Includes TIFF2025061674000115.tif25143.

[0305] Acceptable variations in the sequence of a TGFβRII-DN while retaining its intended function will be known to those of skill in the art. For example, in some embodiments, a dominant-negative receptor of the present invention is a TGFβRII-DN comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 115. In one embodiment, the dominant-negative receptor is a TGFβRII-DN comprising the amino acid sequence set forth in SEQ ID NO: 115.

[0306] In some embodiments, the dominant negative receptor of the present invention is a TGFβRII-DN encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 116. In one embodiment, the dominant negative receptor is a TGFβRII-DN encoded by the nucleic acid sequence set forth in SEQ ID NO: 116.

[0307] In one embodiment, a switch receptor suitable for use in the present invention is the PD1-CTM-CD28 receptor. The PD1-CTM-CD28 receptor converts a negative PD1 signal into a positive CD28 signal when expressed in a cell. The PD1-CTM-CD28 receptor comprises a variant of the PD1 extracellular domain, the CD28 transmembrane domain, and the CD28 cytoplasmic domain. In one embodiment, the PD1-CTM-CD28 receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000116.tif93144: Includes TIFF2025061674000117.tif31144.

[0308] Acceptable variations of the PD1-CTM-CD28 receptor while retaining its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in a cell) will be known to those of skill in the art. Thus, the PD1-CTM-CD28 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PD1-CTM-CD28 receptor set forth in SEQ ID NO:117. Thus, the PD1-CTM-CD28 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the PD1-CTM-CD28 receptor set forth in SEQ ID NO:118.

[0309] In one embodiment, a switch receptor suitable for use in the present invention is a PD1-PTM-CD28 receptor. The PD1-PTM-CD28 receptor converts a negative PD1 signal into a positive CD28 signal when expressed in a cell. The PD1-PTM-CD28 receptor comprises a variant of the PD1 extracellular domain, the PD1 transmembrane domain, and the CD28 cytoplasmic domain. In one embodiment, the PD1-PTM-CD28 receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000118.tif93144: Includes TIFF2025061674000119.tif31144.

[0310] Acceptable variations of the PD1-PTM-CD28 receptor while retaining its intended biological activity (e.g., converting a negative PD1 signal into a positive CD28 signal when expressed in a cell) will be known to those of skill in the art. Thus, the PD1-PTM-CD28 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PD1-PTM-CD28 receptor set forth in SEQ ID NO:119. Thus, the PD1-PTM-CD28 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the PD1-PTM-CD28 receptor set forth in SEQ ID NO:120.

[0311] In one aspect, a switch receptor suitable for use in the present invention is PD1 A132L -PTM-CD28 receptor. PD1 A132LThe -PTM-CD28 receptor converts negative PD1 signals into positive CD28 signals when expressed in cells. A point mutation (A132L) substituting alanine with leucine at amino acid position 132 of PD1 was found to increase its affinity for PD-L1 by two-fold (see, e.g., Zhang et al., Immunity (2004) 20(3), 337-347). PD1 A132L -PTM-CD28 receptor comprises a variant of the PD1 extracellular domain with an amino acid substitution at position 132 (A132L), the PD1 transmembrane domain, and the CD28 cytoplasmic domain. A132L - PTM-CD28 receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000120.tif93144: Includes TIFF2025061674000121.tif31144.

[0312] PD1 while preserving its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in cells). A132L Acceptable variations of the -PTM-CD28 receptor will be known to those of skill in the art. A132L -PTM-CD28 receptor is PD1 as shown in SEQ ID NO: 121 A132L -PTM-A CD28 receptor. A132L -PTM-CD28 receptor is PD1 as shown in SEQ ID NO: 122A132L -PTM- It can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a nucleic acid sequence of a CD28 receptor.

[0313] In one embodiment, the switch receptor suitable for use in the present invention is the PD1-4-1BB receptor. The PD1-4-1BB receptor (also referred to herein as PD1-BB) converts a negative PD1 signal into a positive 4-1BB signal when expressed in a cell. In one embodiment, the PD1-4-1BB receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000122.tif93144: Includes TIFF2025061674000123.tif31142.

[0314] Acceptable variations of the PD1-4-1BB receptor while retaining its intended biological activity (e.g., converting a negative PD1 signal to a positive 4-1BB signal when expressed in a cell) will be known to those of skill in the art. Thus, the PD1-4-1BB receptor of the present invention can include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the PD1-4-1BB receptor set forth in SEQ ID NO:213. Thus, the PD1-4-1BB receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the PD1-4-1BB receptor set forth in SEQ ID NO:214.

[0315] In one aspect, a switch receptor suitable for use in the present invention is PD1 A132L -4-1BB receptor. PD1 A132L The PD1-4-1BB receptor (also referred to herein as PD1*BB) converts a negative PD1 signal into a positive 4-1BB signal when expressed in a cell. A132L The -4-1BB receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000124.tif93144: Includes TIFF2025061674000125.tif31144.

[0316] PD1 while preserving its intended biological activity (e.g., converting a negative PD1 signal to a positive 4-1BB signal when expressed in cells). A132L Acceptable variations of the PD1-4-1BB receptor will be known to those skilled in the art. A132L The PD1 -4-1BB receptor is shown in SEQ ID NO:215 A132L The PD1 receptor of the present invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the PD1 receptor. A132L The PD1 -4-1BB receptor is shown in SEQ ID NO:216 A132L The nucleic acid may be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the -4-1BB receptor.

[0317] In one embodiment, a switch receptor suitable for use in the present invention is the TGFβR-IL12Rβ1 receptor, which converts a negative TGF-β signal into a positive IL-12 signal when expressed in a cell. In one embodiment, the TGFβR-IL12Rβ1 receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000126.tif94145: Includes TIFF2025061674000127.tif32144.

[0318] Acceptable variations of the TGFβR-IL12Rβ1 receptor while retaining its intended biological activity (e.g., converting a negative TGF-β signal into a positive IL-12 signal when expressed in a cell) will be known to those of skill in the art. Thus, the TGFβR-IL12Rβ1 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TGFβR-IL12Rβ1 receptor set forth in SEQ ID NO:123. Thus, the TGFβR-IL12Rβ1 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TGFβR-IL12Rβ1 receptor set forth in SEQ ID NO:124.

[0319] In one embodiment, a switch receptor suitable for use in the present invention is the TGFβR-IL12Rβ2 receptor, which converts a negative TGF-β signal into a positive IL-12 signal when expressed in a cell. In one embodiment, the TGFβR-IL12Rβ2 receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000128.tif162144: Includes TIFF2025061674000129.tif53144.

[0320] Acceptable variations of the TGFβR-IL12Rβ2 receptor while retaining its intended biological activity (e.g., converting a negative TGF-β signal into a positive IL-12 signal when expressed in a cell) will be known to those of skill in the art. Thus, the TGFβR-IL12Rβ2 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TGFβR-IL12Rβ2 receptor set forth in SEQ ID NO:125. Thus, the TGFβR-IL12Rβ2 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TGFβR-IL12Rβ2 receptor set forth in SEQ ID NO:126.

[0321] In one embodiment, a switch receptor suitable for use in the present invention is the TIM3-CD28 receptor, which converts a negative TIM-3 signal into a positive CD28 signal when expressed in a cell. In one embodiment, the TIM3-CD28 receptor has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000130.tif107144: Includes TIFF2025061674000131.tif38144.

[0322] Acceptable variations of the TIM3-CD28 receptor while retaining its intended biological activity (e.g., converting a negative TIM-3 signal to a positive CD28 signal when expressed in a cell) will be known to those of skill in the art. Thus, the TIM3-CD28 receptor of the present invention can comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TIM3-CD28 receptor set forth in SEQ ID NO:127. Thus, the TIM3-CD28 receptor of the present invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TIM3-CD28 receptor shown in SEQ ID NO:128.

[0323] Other dominant-negative receptors and switch receptors suitable for use in the present invention are described in PCT Publication No. WO2013019615A2, the disclosure of which is incorporated herein by reference.

[0324] D. Bispecific antibodies The present invention provides compositions and methods for engineered immune cells or their precursor cells, e.g., engineered T cells, containing nucleic acids encoding bispecific antibodies. Thus, in some embodiments, immune cells are genetically engineered to express bispecific antibodies. As used herein, "bispecific antibodies" refer to antibodies that have binding specificities for at least two different antigen epitopes. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitopes are derived from two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced using coexpression of two immunoglobulin heavy / light chain pairs. See, e.g., Milstein et al. (1983) Nature 305:537-39. Alternatively, bispecific antibodies can be prepared using chemical conjugation. See, e.g., Brennan et al. (1985) Science 229:81. Bispecific antibodies include bispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-48, Gruber et al. (1994) J. Immunol. 152:5368.

[0325] In certain embodiments, the modified cells of the present invention comprise a CAR and a bispecific antibody that has affinity for prostate-specific membrane antigen (PSMA) on a target cell. In certain embodiments, the modified cells of the present invention secrete the bispecific antibody.

[0326] In one embodiment, a bispecific antibody comprises a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen. In some embodiments, the bispecific antibody comprises antigen-binding domains that comprise first and second single-chain variable fragment (scFv) molecules. In one embodiment, the first and second antigen-binding domains bind to an antigen on a target cell and an antigen on an activated T cell.

[0327] In one embodiment, the bispecific antibody comprises specificity for at least one antigen on activated T cells. Activating T cell antigens include antigens found on the surface of T cells that can activate other cells. Activating T cell antigens may bind to costimulatory molecules. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to antigens. Examples of activating T cell antigens can include, but are not limited to, CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or any fragment thereof. In some embodiments, the bispecific antibody comprises specificity for the T cell antigen CD28.

[0328] Other costimulatory elements are also within the scope of the present invention. In these instances, the bispecific antibody recognizes a T cell antigen and may be referred to as a bispecific T cell engager (BiTE). However, the present invention is not limited to the use of any particular bispecific antibody. Rather, any bispecific antibody or BiTE may be used. Bispecific antibody or BiTE molecules may also be expressed as soluble proteins with specificity for at least one target cell-associated antigen.

[0329] In one embodiment, the bispecific antibody comprises more than one antigen-binding domain. In this embodiment, at least one antigen-binding domain comprises a synthetic antibody, a human antibody, a humanized antibody, a single-chain variable fragment, a single-domain antibody, an antigen-binding fragment thereof, and any combination thereof. Techniques for producing human and humanized antibodies are described elsewhere herein.

[0330] In some embodiments, a bispecific antibody comprises more than one antigen-binding domain, wherein at least one antigen-binding domain binds to a negative signaling molecule (e.g., a negative signaling molecule that may be found in the microenvironment of the cell secreting the bispecific antibody) or its interaction partner (e.g., a receptor). In some embodiments, at least one antigen-binding domain of the bispecific antibody binds to TGF-β or its interaction partner (e.g., a receptor). In some embodiments, at least one antigen-binding domain of the bispecific antibody binds to PD-1 or its interaction partner. In one embodiment, at least one antigen-binding domain of the bispecific antibody binds to TGF-βR. In another embodiment, at least one antigen-binding domain of the bispecific antibody binds to PD-L1.

[0331] In some embodiments, a bispecific antibody comprises at least one antigen-binding domain that binds to a molecule on a T cell and activates the T cell. For example, a bispecific antibody of the present disclosure may comprise a superagonist anti-CD28 binding domain as described in U.S. Patent No. 7,585,960, the contents of which are incorporated herein in its entirety.

[0332] In some embodiments, a bispecific antibody comprises at least one antigen-binding domain that binds to PD-L1. For example, a bispecific antibody of the present disclosure may comprise a PD-L1-binding domain derived from 10A5, 13G4, or 1B12, such as, but not limited to, those described in PCT Publication No. WO2007005874A2, the contents of which are incorporated herein in their entirety. In some embodiments, a bispecific antibody comprises at least one antigen-binding domain that binds to a TGF-β receptor, e.g., TGFβRII. For example, a bispecific antibody of the present disclosure may comprise a TGFβRII-binding domain derived from TGF1 or TGF3, such as, but not limited to, those described in U.S. Patent No. 8,147,834, the contents of which are incorporated herein in their entirety.

[0333] Thus, in one embodiment, a bispecific antibody of the disclosure comprises at least one antigen-binding domain that binds PD-L1 or TGFβRII, and an antigen-binding domain that binds CD28.

[0334] In some embodiments, the target cell antigen may be the same antigen as that bound by the T cell receptor, or may be a different antigen. Target cell antigens include any tumor-associated antigen (TAA) or viral, bacterial, and parasitic antigens, or any fragment thereof. Target cell antigens may include any type of ligand that defines the target cell. For example, the target cell antigen may be selected to recognize a ligand that acts as a cellular marker on target cells associated with a particular disease state. Thus, a cellular marker may act as a ligand for the antigen-binding domain in a bispecific antibody, including those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0335] In some embodiments, the target cell antigen is the same antigen as an activating T cell antigen, including, but not limited to, CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and fragments thereof. In one aspect, the invention includes nucleic acids encoding bispecific antibodies comprising dual specificities for an antigen on a target cell and an antigen on an activated T cell, wherein the T cell transiently secretes the bispecific antibody. Techniques for engineering and expressing bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, e.g., Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al, EMBO J. 10: 3655 (1991)), and the "knob-in-hole" technique (see, e.g., U.S. Pat. No. 5,731,168).Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994); and may be made by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991). Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, US2006 / 0025576A1). Bispecific antibodies can be constructed by linking two different antibodies or portions thereof. For example, bispecific antibodies can include Fab, F(ab')2, Fab', scFv, and sdAb derived from two different antibodies.

[0336] Bispecific antibodies of the invention include bispecific antibodies that have affinity for PD-L1 and CD28. In one aspect, the 13G4-1211 PD-L1 / CD28 bispecific antibody of the invention has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000132.tif197144: Includes TIFF2025061674000133.tif66144.

[0337] Permissible variations of the 13G4-1211 PD-L1 / CD28 bispecific antibody that retain its intended biological activity (e.g., binding to PD-L1 and CD28) will be known to those of skill in the art. Thus, the 13G4-1211 PD-L1 / CD28 bispecific antibody of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the 13G4-1211 PD-L1 / CD28 bispecific antibody shown in SEQ ID NO:129. Thus, the 13G4-1211 PD-L1 / CD28 bispecific antibody of the invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the 13G4-1211 PD-L1 / CD28 bispecific antibody shown in SEQ ID NO:130.

[0338] Bispecific antibodies of the invention include bispecific antibodies that have affinity for PD-L1 and CD28. In one aspect, the 10A5-1412 PD-L1 / CD28 bispecific antibody of the invention has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000134.tif196144: Includes TIFF2025061674000135.tif67144.

[0339] Permissible variations of the 10A5-1412 PD-L1 / CD28 bispecific antibody that retain its intended biological activity (e.g., binding to PD-L1 and CD28) will be known to those of skill in the art. Thus, the 10A5-1412 PD-L1 / CD28 bispecific antibody of the invention may comprise an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the 10A5-1412 PD-L1 / CD28 bispecific antibody set forth in SEQ ID NO:131. Thus, the 10A5-1412 PD-L1 / CD28 bispecific antibody of the invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the 10A5-1412 PD-L1 / CD28 bispecific antibody shown in SEQ ID NO:132.

[0340] Bispecific antibodies of the invention include bispecific antibodies that have affinity for PD-L1 and CD28. In one aspect, the 1B12-1412 PD-L1 / CD28 bispecific antibody of the invention has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000136.tif203144: Includes TIFF2025061674000137.tif66144.

[0341] Permissible variations of the 1B12-1412 PD-L1 / CD28 bispecific antibody that retain its intended biological activity (e.g., binding to PD-L1 and CD28) will be known to those of skill in the art. Thus, a 1B12-1412 PD-L1 / CD28 bispecific antibody of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the 1B12-1412 PD-L1 / CD28 bispecific antibody shown in SEQ ID NO:133. Thus, a 1B12-1412 PD-L1 / CD28 bispecific antibody of the invention can be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the 1B12-1412 PD-L1 / CD28 bispecific antibody shown in SEQ ID NO:134.

[0342] Bispecific antibodies of the invention include bispecific antibodies having affinity for TGF-β receptor type II (TGFβRII) and CD28. In one embodiment, the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody of the invention has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000138.tif203145: Includes TIFF2025061674000139.tif66144.

[0343] Acceptable variations of the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody that retain its intended biological activity (eg, binding to TGFβRII and CD28) will be known to those of skill in the art. Thus, a TGFβR-1-1412 TGFβRII / CD28 bispecific antibody of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody shown in SEQ ID NO:135. Thus, the TGFβR-1-1412 TGFβRII / CD28 bispecific antibodies of the invention may be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody shown in SEQ ID NO:136.

[0344] Bispecific antibodies of the invention include bispecific antibodies having affinity for TGF-β receptor type II (TGFβRII) and CD28. In one embodiment, the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody of the invention has the nucleic acid sequence shown below: The amino acid sequence shown below, which can be encoded by TIFF2025061674000140.tif203145: Includes TIFF2025061674000141.tif66144.

[0345] Acceptable variations of the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody that retain its intended biological activity (eg, binding to TGFβRII and CD28) will be known to those of skill in the art. Thus, the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody shown in SEQ ID NO:137. Thus, the TGFβR-3-1412 TGFβRII / CD28 bispecific antibodies of the invention may be encoded by a nucleic acid comprising a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody shown in SEQ ID NO:138.

[0346] Other suitable bispecific antibodies for use in the present invention are described in PCT Publication No. WO2016122738A1, the disclosure of which is incorporated herein by reference.

[0347] E. Nucleic Acids and Expression Vectors The present invention provides nucleic acids encoding CARs and / or dominant-negative receptors and / or switch receptors. In one embodiment, the nucleic acids of the present disclosure comprise nucleic acid sequences encoding the subject CARs of the present invention (e.g., PSMA-CARs). In one embodiment, the nucleic acids of the present disclosure comprise nucleic acid sequences encoding dominant-negative receptors and / or switch receptors (e.g., PD1-PTM-CD28 receptors).

[0348] In some embodiments, the nucleic acids of the present disclosure provide for the production of a CAR and / or a dominant negative receptor and / or a switch receptor, e.g., as described herein, in a mammalian cell. In some embodiments, the nucleic acids of the present disclosure provide for the amplification of nucleic acids encoding a CAR and / or a dominant negative receptor and / or a switch receptor.

[0349] As described herein, the target CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.Therefore, the present disclosure provides a nucleic acid that encodes the antigen binding domain, the transmembrane domain, and the intracellular domain of the target CAR.As described herein, various dominant negative receptors and switch receptors are provided.Therefore, the present invention provides a nucleic acid that encodes a dominant negative receptor and / or a switch receptor.

[0350] In some embodiments, the nucleic acid encoding the CAR is separate from the nucleic acid encoding the dominant negative receptor and / or the switch receptor. In exemplary embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding the dominant negative receptor and / or the switch receptor are present within the same nucleic acid.

[0351] In some embodiments, nucleic acids of the present invention include nucleic acids comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor. In some embodiments, nucleic acids of the present invention include nucleic acids comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor, separated by a linker. Linkers for use in the present invention (e.g., in the context of linking a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor) allow multiple proteins to be encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), and the proteins to be translated as a polyprotein that is dissociated into separate protein components. For example, linkers for use in nucleic acids of the present disclosure comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor allow the CAR and the dominant-negative receptor and / or switch receptor to be translated as a polyprotein that is dissociated into separate CAR and dominant-negative receptor and / or switch receptor components.

[0352] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a start codon, such as ATG, of a protein-coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs available from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select a suitable IRES for use in the present invention.

[0353] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows encoding of multiple proteins as a polyprotein that dissociates into component proteins upon translation. The use of the term "self-cleaving" is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), zosea siagnavirus (TaV), and porcine teschovirus-1 (PTV-1); and cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those of skill in the art will be able to select a suitable self-cleaving peptide for use in the present invention.

[0354] In some embodiments, the nucleic acid of the present disclosure comprises a nucleic acid sequence comprising a coding sequence for a CAR and a coding sequence for a dominant negative receptor and / or a switch receptor, separated by a linker comprising a T2A peptide sequence. In some embodiments, the T2A peptide sequence is TIFF2025061674000142.tif11144. In some embodiments, the linker comprising the T2A peptide sequence may further comprise a spacer sequence as described herein. For example, the linker comprising the T2A peptide sequence may comprise the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 139), which may be encoded by the nucleic acid sequence It may further comprise a spacer sequence comprising the amino acid sequence SGRSGGG (SEQ ID NO: 141), which may be encoded by TIFF2025061674000143.tif4128.

[0355] In some embodiments, the nucleic acids of the present disclosure comprise a nucleic acid sequence comprising a coding sequence for a CAR and a coding sequence for a dominant negative receptor and / or a switch receptor, separated by a linker comprising an F2A peptide sequence. In some embodiments, the F2A peptide sequence is An amino acid sequence that can be encoded by TIFF2025061674000144.tif11144 Includes TIFF2025061674000145.tif4128.

[0356] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors prior to their secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including, but not limited to, Arg-X-Lys-Arg (SEQ ID NO:145) or Arg-X-Arg-Arg (SEQ ID NO:146), such as Arg-Gln-Lys-Arg (SEQ ID NO:148), and Arg-XX-Arg (SEQ ID NO:147) (wherein X is any naturally occurring amino acid). Another example of a furin cleavage site is X1-Arg-X2-X3-Arg (SEQ ID NO:149), where X1 is Lys or Arg, X2 is any naturally occurring amino acid, and X3 is Lys or Arg. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0357] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding furin and F2A, a linker comprising a nucleic acid sequence encoding furin and E2A, a linker comprising a nucleic acid sequence encoding furin and P2A, or a linker comprising a nucleic acid sequence encoding furin and T2A. Those skilled in the art will be able to select a suitable combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between furin and the 2A peptide. A variety of spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO:1), and (GGGS)n (SEQ ID NO:2), where n represents an integer of at least 1. Exemplary spacer sequences include, but are not limited to: TIFF2025061674000146.tif18137, etc. Those skilled in the art will be able to select suitable spacer sequences for use in the present invention.

[0358] In some embodiments, the nucleic acids of the present disclosure comprise a nucleic acid sequence comprising a coding sequence for a CAR and a coding sequence for a dominant-negative receptor and / or a switch receptor, separated by a furin-(G4S)2-T2A (F-GS2-T2A) linker. The F-GS2-T2A linker is a nucleic acid sequence TIFF2025061674000147.tif18143, and the amino acid sequence TIFF2025061674000148.tif4128. One of skill in the art will recognize that the linkers of the present invention may include sequence variations that are tolerated.

[0359] In some embodiments, the present invention provides a nucleic acid comprising a nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor as described herein. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and a nucleic acid sequence encoding a CAR (e.g., a PSMA-CAR) as described herein. In one embodiment, the nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and the nucleic acid sequence encoding the CAR are present on separate nucleic acids. In one embodiment, the nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and the nucleic acid sequence encoding the CAR are present in the same nucleic acid. In such embodiments, the nucleic acid sequence encoding the dominant-negative receptor and / or a switch receptor and the nucleic acid sequence encoding the CAR are separated by a linker as described herein.

[0360] For example, a nucleic acid of the present disclosure may comprise a nucleic acid sequence encoding a dominant receptor, a linker, and a nucleic acid sequence encoding a CAR. In one embodiment, the linker comprises a nucleic acid sequence encoding a 2A peptide (e.g., T2A). In an exemplary embodiment, a nucleic acid of the present disclosure may comprise a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor and a nucleic acid sequence encoding a CAR, separated by a linker sequence comprising a nucleic acid sequence encoding T2A.

[0361] Thus, in one embodiment, a nucleic acid of the disclosure comprises, in the 5' to 3' direction: a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor, a nucleic acid sequence encoding a linker, and a nucleic acid sequence encoding a CAR. In one embodiment, a nucleic acid of the disclosure comprises, in the 5' to 3' direction: a nucleic acid sequence encoding a CAR, a nucleic acid sequence encoding a linker, and a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor.

[0362] In some embodiments, a nucleic acid of the present disclosure comprises, in the 5' to 3' direction: a nucleic acid sequence encoding a dominant-negative receptor and / or a switch receptor, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the dominant-negative receptor is TGFβRII-DN. In one embodiment, the CAR is mouse J591 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the present invention comprises, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding mouse J591 PSMA-CAR. In one embodiment, a nucleic acid comprising, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding mouse J591 PSMA-CAR, is Includes TIFF2025061674000149.tif31144TIFF2025061674000150.tif230144TIFF2025061674000151.tif18142.

[0363] In one embodiment, the CAR is a humanized J591 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5' to 3': a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence encoding a humanized J591 PSMA-CAR. In one embodiment, a nucleic acid of the disclosure comprises, from 5' to 3': a nucleic acid encoding a humanized PSMA-CAR, a nucleic acid encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid encoding a dominant-negative receptor and / or a switch receptor.

[0364] In one embodiment, the CAR is a humanized J591 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5' to 3': a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a humanized J591 PSMA-CAR. In one embodiment, the nucleic acid comprises, from 5' to 3': a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a humanized J591 PSMA-CAR.

[0365] A humanized PSMA-CAR can comprise any of the heavy chain variable regions and light chain variable regions disclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1. For example, a humanized PSMA-CAR of the present invention can comprise an scFv comprising any of the heavy chain variable regions and light chain variable regions disclosed therein. In some embodiments, a humanized J591 PSMA-CAR comprises a humanized J591 PSMA-binding domain comprising a heavy chain variable region and a light chain variable region selected from any of the heavy chain variable region sequences and light chain variable region sequences shown in Table 19.

[0366] In some embodiments, a nucleic acid of the invention comprising, in 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence encoding humanized J591 PSMA-CAR, is represented by the following nucleic acid sequence: Includes TIFF2025061674000152.tif176145.

[0367] In some embodiments, a nucleic acid of the invention comprising, in 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence encoding humanized J591 PSMA-CAR, is represented by the following nucleic acid sequence: Includes TIFF2025061674000153.tif176144.

[0368] In some embodiments, a nucleic acid of the invention comprising, in 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence encoding humanized J591 PSMA-CAR, is represented by the following nucleic acid sequence: Includes TIFF2025061674000154.tif176145.

[0369] In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR. In one embodiment, the nucleic acid comprising, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR, is the nucleic acid sequence shown below: Includes TIFF2025061674000155.tif79144 and TIFF2025061674000156.tif203144.

[0370] In one embodiment, the CAR is a human 2A10 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR. In one embodiment, a nucleic acid comprising, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR, is a nucleic acid comprising the nucleic acid sequence shown below: Includes TIFF2025061674000157.tif203144 and TIFF2025061674000158.tif80145.

[0371] In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR. In one embodiment, the nucleic acid comprising, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR, is the nucleic acid sequence shown below: Includes TIFF2025061674000159.tif93144 and TIFF2025061674000160.tif189144.

[0372] In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR. In one embodiment, the nucleic acid comprising, in the 5' to 3' direction: a nucleic acid sequence encoding TGFβRII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR, is the nucleic acid sequence shown below: Includes TIFF2025061674000161.tif216144 and TIFF2025061674000162.tif66143.

[0373] Acceptable variations in the nucleic acid sequences encoding TGFβRII-DN and PSMA-CAR will be known to those of skill in the art. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 152-156, 258, 260, or 262. In one embodiment, the nucleic acid sequence encoding TGFβRII-DN and mouse J591 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 152. In one embodiment, the nucleic acid sequence encoding the TGFβRII-DN and human 1C3 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 153. In one embodiment, the nucleic acid sequence encoding the TGFβRII-DN and human 2A10 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 154. In one embodiment, the nucleic acid sequence encoding the TGFβRII-DN and human 2F5 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 155. In one embodiment, the nucleic acid sequence encoding the TGFβRII-DN and human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 156. In one embodiment, the nucleic acid sequence encoding the TGFβRII-DN and humanized J591 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 258, 260, or 262.

[0374] In some embodiments, a nucleic acid of the disclosure comprises, in 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD28.

[0375] In one aspect, the CAR is a humanized J591 CAR (huJ591 PSMA-CAR). Thus, in an exemplary aspect, a nucleic acid of the invention comprises, from 5' to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker (e.g., a 2A linker), and a nucleic acid sequence encoding a huJ591 PSMA-CAR. In some embodiments, a nucleic acid of the invention comprises, from 5' to 3': a nucleic acid sequence encoding PD1-CTM-CD28 having the amino acid sequence set forth in SEQ ID NO:117, a nucleic acid sequence encoding a linker (e.g., a 2A linker), and a nucleic acid sequence encoding a huJ591 PSMA-CAR having the amino acid sequence set forth in SEQ ID NO:245 or 247.

[0376] In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, in the 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR. In one embodiment, the nucleic acid comprising, in the 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR, is the nucleic acid sequence shown below: Includes TIFF2025061674000163.tif113144 and TIFF2025061674000164.tif183144.

[0377] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD28. In one embodiment, the CAR is a human 2A10 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, in a 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR. In one embodiment, a nucleic acid comprising, in a 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR, is Includes TIFF2025061674000165.tif196144 and TIFF2025061674000166.tif93144.

[0378] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD28. In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, in a 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR. In one embodiment, a nucleic acid comprising, in a 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR, is Includes TIFF2025061674000167.tif58144TIFF2025061674000168.tif223144TIFF2025061674000169.tif11140.

[0379] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD28. In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, in a 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR. In one embodiment, a nucleic acid comprising, in a 5' to 3' direction: a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR, is Includes TIFF2025061674000170.tif141144 and TIFF2025061674000171.tif155144.

[0380] Acceptable variations in the nucleic acid sequences encoding PD1-CTM-CD28 and PSMA-CAR will be known to those of skill in the art. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs:157-160. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 1C3 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO:157. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2A10 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 158. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2F5 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 159. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 160.

[0381] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in a 5' to 3' direction: PD1 A132L -PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR. In one embodiment, in the 5' to 3' direction: PD1 A132L- The nucleic acid comprising a nucleic acid sequence encoding PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 1C3 PSMA-CAR is selected from the nucleic acid sequence shown below: Includes TIFF2025061674000172.tif113144 and TIFF2025061674000173.tif176144.

[0382] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28.

[0383] In one embodiment, the CAR is a humanized J591 CAR (huJ591 PSMA-CAR). Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in 5' to 3' direction: PD1 A132L In some embodiments, the nucleic acid of the invention comprises a nucleic acid sequence encoding -PTM-CD28, a nucleic acid sequence encoding a linker (e.g., a 2A linker), and a nucleic acid sequence encoding huJ591 PSMA-CAR. In some embodiments, the nucleic acid of the invention comprises a PD1 PSMA-CAR having, in a 5' to 3' direction, the amino acid sequence set forth in SEQ ID NO:121. A132L -PTM-CD28, a nucleic acid sequence encoding a linker (e.g., a 2A linker), and a nucleic acid sequence encoding a huJ591 PSMA-CAR having the amino acid sequence shown in SEQ ID NO:245 or 247.

[0384] In one embodiment, the CAR is a human 2A10 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in 5' to 3' direction: PD1 A132L -PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR. In one embodiment, in the 5' to 3' direction: PD1 A132L- The nucleic acid comprising a nucleic acid sequence encoding PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2A10 PSMA-CAR is selected from the nucleic acid sequence shown below: Includes TIFF2025061674000174.tif141144 and TIFF2025061674000175.tif148144.

[0385] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in a 5' to 3' direction: PD1 A132L -PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR. In one embodiment, in the 5' to 3' direction: PD1 A132L - The nucleic acid comprising a nucleic acid sequence encoding PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR is selected from the nucleic acid sequence shown below: Includes TIFF2025061674000176.tif230144 and TIFF2025061674000177.tif59144.

[0386] In some embodiments, a nucleic acid of the disclosure comprises, in a 5' to 3' direction: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the invention comprises, in a 5' to 3' direction: PD1 A132L-PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR. In one embodiment, in the 5' to 3' direction: PD1 A132L - The nucleic acid comprising a nucleic acid sequence encoding PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2C6 PSMA-CAR is selected from the nucleic acid sequence shown below: Includes TIFF2025061674000178.tif86144TIFF2025061674000179.tif203144.

[0387] PD1 A132L -Permissible variations in nucleic acid sequences encoding PTM-CD28 and PSMA-CAR will be known to those of skill in the art. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 161-164. In one embodiment, PD1 A132L - The nucleic acid sequence encoding PTM-CD28 and human 1C3 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 161. A132L - The nucleic acid sequence encoding PTM-CD28 and human 2A10 PSMA-CAR comprises the nucleic acid sequence shown in SEQ ID NO: 162. A132L - The nucleic acid sequence encoding PTM-CD28 and human 2F5 PSMA-CAR co...

Claims

1. (a) a chimeric antigen receptor (CAR) capable of binding to prostate-specific membrane antigen (PSMA), comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain comprises a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 183; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 184; (b) dominant-negative receptors and / or switch receptors; A modified immune cell or a precursor thereof, comprising:

2. The modified cell of claim 1, wherein VH comprises the sequence of SEQ ID NO:

191.

3. The modified cell of claim 1 or 2, wherein the VL comprises the sequence of SEQ ID NO:

192.

4. The modified cell of any one of claims 1 to 3, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

5. The modified cell of claim 4, wherein the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single domain antibody.

6. The modified cell of claim 1, wherein the transmembrane domain comprises a transmembrane region derived from CD8.

7. The modified cell of claim 6, wherein the transmembrane region derived from CD8 comprises the amino acid sequence shown in SEQ ID NO:

88.

8. The modified cell of claim 6 or 7, wherein the transmembrane domain further comprises a hinge region derived from CD8.

9. The modified cell of claim 8, wherein the hinge region derived from CD8 comprises the amino acid sequence shown in SEQ ID NO:

86.

10. The modified cell of any one of claims 6 to 9, wherein the intracellular domain comprises a 4-1BB signaling domain and a CD3 zeta signaling domain.

11. The modified cell of any one of claims 6 to 10, wherein the intracellular domain comprises an ICOS signaling domain and a CD3 zeta signaling domain.

12. The modified cell of any one of claims 6 to 11, wherein the intracellular domain comprises a variant ICOS signaling domain and a CD3 zeta signaling domain.

13. The modified cell of claim 10, wherein the 4-1BB signaling domain comprises the amino acid sequence shown in SEQ ID NO:

92.

14. The modified cell of claim 11, wherein the ICOS signaling domain comprises the amino acid sequence shown in SEQ ID NO:

203.

15. The modified cell of claim 12, wherein the variant ICOS signaling domain comprises the amino acid sequence shown in SEQ ID NO:

95.

16. The modified cell of any one of claims 10 to 15, wherein the CD3 zeta signaling domain comprises the amino acid sequence shown in SEQ ID NO: 97 or 100.

17. The modified cell of any one of the preceding claims, wherein the dominant negative receptor is a truncated variant of a wild-type protein associated with negative signaling.

18. The modified cell of claim 17, wherein the truncated variant of the wild-type protein associated with negative signals comprises the amino acid sequence shown in SEQ ID NO:

115.

19. 19. The modified cell of any one of claims 1-18, wherein the switch receptor comprises a first domain derived from a first polypeptide associated with a negative signal; and a second domain derived from a second polypeptide associated with a positive signal.

20. The modified cell of claim 19, wherein the first domain comprises at least a portion of an extracellular domain of a first polypeptide associated with a negative signal, and the second domain comprises at least a portion of an intracellular domain of a second polypeptide associated with a positive signal.

21. 3. The modified cell of any one of the preceding claims, wherein the switch receptor further comprises a transmembrane domain of the switch receptor.

22. 22. The modified cell of claim 21, wherein the transmembrane domain of the switch receptor comprises a transmembrane domain of a first polypeptide associated with a negative signal; or a transmembrane domain of a second polypeptide associated with a positive signal.

23. The modified cell of any one of claims 19 to 22, wherein the first polypeptide associated with a negative signal is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR.

24. The modified cell of any one of claims 19 to 23, wherein the second polypeptide associated with a positive signal is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R.

25. 23. The modified cell of any one of claims 19-22, wherein the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of CD28; and a second domain comprising at least a portion of the intracellular domain of CD28.

26. 26. The modified cell of claim 25, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

117.

27. 23. The modified cell of any one of claims 19-22, wherein the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a transmembrane domain of the switch receptor comprising at least a portion of the transmembrane domain of PD1; and a second domain comprising at least a portion of the intracellular domain of CD28.

28. 28. The modified cell of claim 27, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

119.

29. 28. The modified cell of claim 27, wherein the first domain comprises at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132.

30. 30. The modified cell of claim 29, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

121.

31. 23. The modified cell of any one of claims 19-22, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of CD28.

32. 32. The modified cell of claim 31 , wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

121.

33. 22. The modified cell of any one of claims 19-21, wherein the switch receptor comprises: a first domain comprising at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of 4-1BB.

34. 34. The modified cell of claim 33, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO: 2l5.

35. 22. The modified cell of any one of claims 19-21, wherein the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TIM-3; and a second domain comprising at least a portion of the intracellular domain of CD28.

36. 36. The modified cell of claim 35, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

127.

37. 22. The modified cell of any one of claims 19-21, wherein the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rαl.

38. 38. The modified cell of claim 37, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

123.

39. 22. The modified cell of any one of claims 19-21, wherein the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rβ1.

40. 40. The modified cell of claim 39, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:

125.

41. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A dominant negative receptor comprising the amino acid sequence shown in SEQ ID NO:

115. A modified immune cell or a precursor thereof, comprising:

42. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A switch receptor comprising the amino acid sequence shown in SEQ ID NO: 213 or 215. A modified immune cell or a precursor thereof, comprising:

43. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A switch receptor comprising the amino acid sequence shown in SEQ ID NO: 117 or 119. A modified immune cell or a precursor thereof, comprising:

44. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A switch receptor comprising the amino acid sequence shown in SEQ ID NO:

121. A modified immune cell or a precursor thereof, comprising:

45. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A switch receptor comprising the amino acid sequence shown in SEQ ID NO: 127; A modified immune cell or a precursor thereof, comprising:

46. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A switch receptor comprising the amino acid sequence shown in SEQ ID NO: 123; A modified immune cell or a precursor thereof, comprising:

47. A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising a PSMA-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A switch receptor comprising the amino acid sequence shown in SEQ ID NO: 125; A modified immune cell or a precursor thereof, comprising:

48. The modified cell of any one of the preceding claims, which is a modified T cell.

49. 49. The modified T cell of claim 48, which is an autologous cell.

50. 49. The modified T cell of claim 48, which is an allogeneic cell.

51. 51. The modified cell of any one of claims 1 to 50, which is a cytotoxic T lymphocyte (CTL).

52. The modified cell of any one of the preceding claims, which is derived from a human cell.

53. (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to a prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, the antigen-binding domain comprising a heavy chain variable region (VH) comprising the consensus sequence of SEQ ID NO: 183; and a light chain variable region (VL) comprising the consensus sequence of SEQ ID NO: 184; (b) a second nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor; An isolated nucleic acid comprising:

54. 54. The isolated nucleic acid of claim 53, wherein VH comprises the sequence of SEQ ID NO:

191.

55. 55. The isolated nucleic acid of claim 53 or 54, wherein the VL comprises the sequence of SEQ ID NO:

192.

56. 54. The isolated nucleic acid of claim 53, wherein the first nucleic acid sequence comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 246, 248, 250, 252, 254, or 256.

57. 54. The isolated nucleic acid of claim 53, wherein the second nucleic acid sequence comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216.

58. 58. The isolated nucleic acid of any one of claims 53 to 57, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker.

59. 59. The isolated nucleic acid of claim 58, wherein the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES).

60. 59. The isolated nucleic acid of claim 58, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving peptide.

61. 61. The isolated nucleic acid of claim 60, wherein the self-cleaving peptide is a 2A peptide.

62. 62. The isolated nucleic acid of claim 61, wherein the 2A peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), Equine rhinitis A virus 2A (E2A), and Foot and Mouth Disease virus 2A (F2A).

63. 62. The isolated nucleic acid of claim 61, wherein the 2A peptide is T2A.

64. 62. The isolated nucleic acid of claim 61, wherein the 2A peptide is F2A.

65. 65. The isolated nucleic acid of any one of claims 53-64, comprising in the 5' to 3' direction: a first nucleic acid sequence, a linker, and a second nucleic acid sequence.

66. 65. The isolated nucleic acid of any one of claims 53-64, comprising in the 5' to 3' direction: a second nucleic acid sequence, a linker, and the first nucleic acid sequence.

67. a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to a prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, the antigen-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; a second nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216; An isolated nucleic acid comprising:

68. a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding to a prostate-specific membrane antigen (PSMA), the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, the antigen-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A second nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor comprising the nucleic acid sequence shown in SEQ ID NO: 116; An isolated nucleic acid comprising:

69. 69. The isolated nucleic acid of claim 68, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding T2A.

70. 69. The isolated nucleic acid of claim 68, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding F2A.

71. 71. An expression construct comprising the isolated nucleic acid of any one of claims 53 to 70.

72. 72. The expression construct of claim 71, which is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

73. 72. The expression construct of claim 71, which is a lentiviral vector.

74. The expression construct of claim 73, wherein the lentiviral vector further comprises an EF-la promoter.

75. The expression construct of claims 72-74, wherein the lentiviral vector further comprises a rev response element (RRE).

76. 76. The expression construct of any one of claims 72 to 75, wherein the lentiviral vector further comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).

77. The expression construct of any one of claims 72 to 76, wherein the lentiviral vector further comprises a cPPT sequence.

78. 78. The expression construct of claim 77, wherein the lentiviral vector further comprises an EF-la promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence.

79. The expression construct of any one of claims 72 to 78, wherein the lentiviral vector is a self-inactivating lentiviral vector.

80. 8. A method for generating a modified immune cell or a precursor thereof according to any one of claims 1 to 52, comprising introducing into an immune cell one or more of the nucleic acids according to any one of claims 53 to 70 or the expression constructs according to any one of claims 71 to 79.

81. 53. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune cell of any one of claims 1-52.

82. 82. The method of claim 81, further comprising administering lymphodepleting chemotherapy to the subject.

83. 83. The method of claim 82, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and / or fludarabine.

84. 1. A method of treating prostate cancer in a subject in need thereof, the method comprising: administering to the subject lymphodepleting chemotherapy, comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and Administering the modified T cells to a subject. Including, The modified T cell is A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, the antigen-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A dominant negative receptor comprising the amino acid sequence shown in SEQ ID NO:

115. A method comprising:

85. 1. A method of treating metastatic castration-resistant prostate cancer in a subject in need thereof, the method comprising: administering to the subject lymphodepleting chemotherapy, comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and Administering the modified T cells to a subject. Including, The modified T cell is A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, the CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, the antigen-binding domain comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 192; A dominant negative receptor comprising the amino acid sequence shown in SEQ ID NO:

115. A method comprising:

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