Prostate-specific membrane antigen CAR and methods of use thereof

Abstract

The present disclosure provides modified immune cells (e.g., modified T cells) comprising a chimeric antigen receptor (CAR) with affinity for prostate-specific membrane antigen (PSMA) (e.g., human PSMA). The present disclosure provides modified immune cells (e.g., modified T cells) comprising CAR with affinity for PSMA and a dominant negative receptor and / or a conversion receptor. The present disclosure provides modified immune cells (e.g., modified T cells) comprising CAR with affinity for PSMA and a dominant negative receptor and / or a conversion receptor, wherein the modified cells are capable of expressing and secreting bispecific antibodies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Application No. 62 / 639,321, filed on March 6, 2018, which is incorporated herein by reference in its entirety. Background Art

[0003] A major challenge in the application of immunotherapy to solid malignancies is breaking tolerance to self-antigens. Vaccine strategies aimed at harnessing endogenous anti-tumor T cells are limited by the T cell receptor (TCR) repertoire, which can be deleted in the thymus as part of central tolerance or rendered nonfunctional by post-thymic mechanisms of peripheral tolerance. One strategy to overcome this obstacle is to use the chimeric antigen receptor (CAR) approach to generate genetically engineered T cells that are redirected to tumor antigens. 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 the TCR.

[0004] Prostate-specific membrane antigen (PSMA) is a membrane-bound protein expressed on the cell surface and reportedly highly overexpressed in prostate cancer tissue. PSMA expression is directly correlated with tumor grade and stage progression and is thought to confer a selective growth advantage on prostate cancer cells. Therefore, PSMA may be an ideal target for immunotherapy against prostate cancer.

[0005] Another major challenge in cancer immunotherapy is the hostile microenvironment in which the targeted tumor resides. For example, immunosuppressive receptor ligands, such as PDL1 (CD274), which binds to PD1 (CD279), are upregulated in the tumor microenvironment and negatively regulate T cell activity. Furthermore, TGF-β, which is overexpressed in prostate tumor cells, can act as an immunosuppressive molecule.

[0006] Therefore, there is a need in the art for novel cancer immunotherapies targeting PSMA, and the present invention satisfies this need. Summary of the Invention

[0007] The present invention is based on the discovery that human and murine prostate-specific membrane antigen (PSMA) chimeric antigen receptor (CAR) T cells exhibit potent anti-tumor 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 anti-tumor activity.

[0008] Thus, in certain aspects, the present disclosure provides a modified immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain; and a dominant negative receptor and / or a switch receptor.

[0009] In certain exemplary embodiments, the PSMA-binding domain is a murine PSMA-binding domain.

[0010] In certain exemplary embodiments, the PSMA-binding domain is a human PSMA-binding domain.

[0011] In certain exemplary embodiments, the PSMA-binding domain is selected from an antibody, a Fab, or a scFv.

[0012] In certain exemplary embodiments, the scFv comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13, 14, 26, 38, 50, or 62.

[0013] In certain exemplary embodiments, the CAR comprises a transmembrane domain, and an intracellular domain.

[0014] In certain exemplary embodiments, the transmembrane domain comprises a transmembrane region derived from CD8.

[0015] In certain exemplary embodiments, the transmembrane region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO:88.

[0016] In certain exemplary embodiments, the transmembrane domain further comprises a hinge region derived from CD8.

[0017] In certain exemplary embodiments, the hinge region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO:86.

[0018] In certain exemplary embodiments, the intracellular domain comprises a 4-1BB signaling domain and a CD3 zeta signaling domain.

[0019] In certain exemplary embodiments, the intracellular domain comprises an ICOS signaling domain and a CD3 zeta signaling domain.

[0020] In certain exemplary embodiments, the intracellular domain comprises a variant ICOS signaling domain and a CD3 zeta signaling domain.

[0021] In certain exemplary embodiments, the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO:92.

[0022] In certain exemplary embodiments, the ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID NO:203.

[0023] In certain exemplary embodiments, the variant ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID NO:95.

[0024] In certain exemplary embodiments, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO:97 or 100.

[0025] In certain exemplary embodiments, a dominant negative receptor is a truncated variant of a wild-type protein that associates with a negative signal.

[0026] In certain exemplary embodiments, the truncated variant of the wild-type protein associated with the negative signal comprises the amino acid sequence set forth in SEQ ID NO:115.

[0027] In certain exemplary embodiments, the switch receptor comprises a first domain, wherein the first domain is derived from a first polypeptide associated with a negative signal; and a second domain, wherein the second domain is derived from a second polypeptide associated with a positive signal.

[0028] In certain exemplary embodiments, the first domain comprises at least a portion of the extracellular domain of a first polypeptide associated with a negative signal, and wherein the second domain comprises at least a portion of the extracellular domain of a second polypeptide associated with a positive signal.

[0029] In certain exemplary embodiments, the switch receptor further comprises a switch receptor transmembrane domain.

[0030] In certain exemplary embodiments, the switch receptor transmembrane domain comprises the transmembrane domain of a first polypeptide associated with a negative signal; or the transmembrane domain of a second polypeptide associated with a positive signal.

[0031] In certain exemplary embodiments, the first polypeptide associated with a negative signal is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR.

[0032] In certain exemplary embodiments, the second polypeptide associated with a positive signal is selected from CD28, ICOS, 4-1BB, and IL-12R.

[0033] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a switch receptor transmembrane domain 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.

[0034] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:117.

[0035] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1; a switch receptor transmembrane domain 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.

[0036] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:119.

[0037] In certain exemplary embodiments, the first domain includes at least a portion of the extracellular domain of PD1 that contains an alanine (A) to leucine (L) substitution at amino acid position 132.

[0038] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:121.

[0039] In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1, which 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.

[0040] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:121.

[0041] In certain exemplary embodiments, the switch receptor includes a first domain comprising at least a portion of the extracellular domain of PD1, which contains 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.

[0042] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:215.

[0043] 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.

[0044] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:127.

[0045] 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.

[0046] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:123.

[0047] 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 IL12Rβ2.

[0048] In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO:125.

[0049] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13, 14, 16, 38, 50, or 62; and a dominant negative receptor comprising the amino acid sequence recited in SEQ ID NO: 115.

[0050] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13, 14, 16, 38, 50, or 62; and a switch receptor comprising the amino acid sequence recited in SEQ ID NOs: 213 or 215.

[0051] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13, 14, 16, 38, 50, or 62; and a switch receptor comprising the amino acid sequence recited in SEQ ID NOs: 117 or 119.

[0052] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13, 14, 16, 38, 50, or 62; and a switch receptor comprising the amino acid sequence recited in SEQ ID NO: 121.

[0053] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13, 14, 16, 38, 50, or 62; and a switch receptor comprising the amino acid sequence recited in SEQ ID NO: 127.

[0054] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13, 14, 16, 38, 50, or 62; and a switch receptor comprising the amino acid sequence recited in SEQ ID NO: 123.

[0055] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 14, 16, 38, 50, or 62; and a switch receptor comprising the amino acid sequence recited in SEQ ID NO: 125.

[0056] In another aspect, the present disclosure provides a modified immune cell or a precursor cell thereof, comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in any one of SEQ ID NOs: 13 and 14; and a switch receptor comprising the amino acid sequence recited in SEQ ID NO: 115.

[0057] In certain exemplary embodiments, the modified cells secrete bispecific antibodies.

[0058] In certain exemplary embodiments, a bispecific antibody comprises a first antigen-binding domain and a second antigen-binding domain.

[0059] In certain exemplary embodiments, the first antigen binding domain binds to a negative signal selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and TGFβR.

[0060] In certain exemplary embodiments, the second antigen binding domain binds to a co-stimulatory molecule.

[0061] In certain exemplary embodiments, the costimulatory molecule is CD28.

[0062] In certain exemplary embodiments, the modified cell is a modified T cell.

[0063] In certain exemplary embodiments, the modified T cells are autologous cells.

[0064] In certain exemplary embodiments, the modified cells are cytotoxic T lymphocytes (CTLs).

[0065] In certain exemplary embodiments, the modified cells are natural killer (NK) cells.

[0066] In certain exemplary embodiments, the modified cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

[0067] In certain exemplary embodiments, the modified cells are autologous cells.

[0068] In certain exemplary embodiments, the modified cells are of human origin.

[0069] In certain exemplary embodiments, the modified T cells are of human origin.

[0070] In another aspect, the present disclosure provides an isolated nucleic acid comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain; and a second nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor.

[0071] In certain exemplary embodiments, the first nucleic acid sequence comprises a nucleic acid sequence recited in any one of SEQ ID NOs: 106, 108, 110, 112, 114, 210, and 212.

[0072] In certain exemplary embodiments, the second nucleic acid sequence comprises the nucleic acid sequence recited in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216.

[0073] In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker.

[0074] In certain exemplary embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES).

[0075] In certain exemplary embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide.

[0076] In certain exemplary embodiments, the self-cleaving peptide is a 2A peptide.

[0077] In certain exemplary embodiments, the 2A peptide is selected from porcine teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A).

[0078] In certain exemplary embodiments, the 2A peptide is T2A.

[0079] In certain exemplary embodiments, the 2A peptide is F2A.

[0080] In certain exemplary embodiments, the isolated nucleic acid comprises, from 5' to 3', a first nucleic acid sequence, a linker, and a second nucleic acid sequence.

[0081] In certain exemplary embodiments, the isolated nucleic acid comprises, from 5' to 3', the second nucleic acid sequence, the linker, and the first nucleic acid sequence.

[0082] In another aspect, the present disclosure provides an isolated nucleic acid comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising a nucleic acid sequence recited in any one of SEQ ID NOs: 180, 15, 27, 39, 51, or 63; and a second nucleic acid sequence encoding a dominant negative receptor and / or switch receptor comprising a nucleic acid sequence recited in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216.

[0083] In another aspect, the present disclosure provides an isolated nucleic acid comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the nucleic acid sequence recited in SEQ ID NO: 180; and a second nucleic acid sequence encoding a dominant negative receptor and / or switch receptor comprising the nucleic acid sequence recited in SEQ ID NO: 116.

[0084] In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding T2A.

[0085] In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding F2A.

[0086] In another aspect, the present disclosure provides an isolated nucleic acid comprising the nucleic acid sequence recited in any one of SEQ ID NOs: 152-168, 210, 212, and 217-226.

[0087] In another aspect, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding a bispecific antibody as recited in any one of SEQ ID NOs: 130, 132, 134, 136, or 138.

[0088] In another aspect, the present disclosure provides an expression construct comprising the isolated nucleic acid of any one of the above embodiments.

[0089] In certain exemplary embodiments, the expression construct is a viral vector selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0090] In certain exemplary embodiments, the expression construct is a lentiviral vector.

[0091] In certain exemplary embodiments, the lentiviral vector further comprises an EF-1α promoter.

[0092] In certain exemplary embodiments, the lentiviral vector further comprises a rev response element (RRE).

[0093] In certain exemplary embodiments, the lentiviral vector further comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

[0094] In certain exemplary embodiments, the lentiviral vector further comprises a cPPT sequence.

[0095] In certain exemplary embodiments, the lentiviral vector further comprises an EF-1α promoter, a rev response element (RRE), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a cPPT sequence.

[0096] In certain exemplary embodiments, the lentiviral vector is a self-inactivating lentiviral vector.

[0097] In another aspect, the present disclosure provides a method for producing a modified immune cell or a precursor cell thereof according to any of the above embodiments, comprising introducing one or more of the nucleic acid according to any of the above embodiments or the expression construct according to any of the above embodiments into an immune cell.

[0098] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune cell of any one of the above embodiments.

[0099] In certain exemplary embodiments, the method further comprises administering lymphodepleting chemotherapy to the subject.

[0100] In certain exemplary embodiments, the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and / or fludarabine.

[0101] In another aspect, the present disclosure provides a method for treating prostate cancer in a subject in need thereof. The method comprises administering to the subject lymphodepleting chemotherapy comprising a therapeutically effective amount of cyclophosphamide and a modified T cell comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence recited in SEQ ID NO: 13; and a dominant negative receptor comprising the amino acid sequence recited in SEQ ID NO: 115.

[0102] 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 a modified T cell comprising a chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] 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.It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0104] Figure 1AResults using purified IVT PSMA RNA CAR and full-length PSMA RNA resolved on agarose gel are illustrated.

[0105] Figure 1B Figure 2 shows the results of electroporating purified PSMA RNA CAR into ND444 T cells and examining CAR expression by flow cytometry. The mean fluorescence intensity is marked below the graph.

[0106] Figure 1C PSMA expression is shown. Purified full-length PSMA RNA was electroporated into Nalm6 or K562 cells (center and right panels). PSMA expression was examined by flow cytometry.

[0107] Figure 1D Graph illustrating the results of using combined PC3.PSMA single cell cloning. PC3.PSMA cells were subjected to limiting dilution (left panel), and seven individual colonies were isolated and pooled into a new cell line, PC3.PSMA.7SC (right panel). PSMA expression was examined by flow cytometry.

[0108] Figure 2A Graph illustrating the results of incubating tumor cells with various PSMA RNA CARs and performing a CD107a assay. Cells were gated by CD3.

[0109] Figure 2B Graph showing the results of luciferase-based CTL assays using various PSMA RNA CARs cultured with tumor cells. Results are reported as percent killing based on luciferase activity in wells with tumor alone in the absence of T cells.

[0110] Figure 2C The results of ELISA assays using various PSMA RNA CARs co-cultured with tumor cells are shown (IL-2, left panel; IFN-γ, right panel).

[0111] Figure 3A Graphs show results using a PSMA Lenti CAR constructed and transduced into primary human T cells (MOI=3). CAR expression was examined by flow cytometry on day 8.

[0112] Figure 3B Results are shown using various PSMA Lenti CARs cultured with or without tumor cells and assayed for CD107a. Cells were gated by CD3. Results from day 12 are shown.

[0113] Figure 3CResults are shown for a luciferase-based CTL assay using various PSMA Lenti CARs cultured with or without tumor cells. Results are reported as percent killing based on luciferase activity in wells harboring tumor alone in the absence of T cells. Results from day 12 are shown.

[0114] Figure 3D Graph showing the results of ELISA assays (IL-2, left panel; IFN-γ, right panel) using various PSMA Lenti CARs co-cultured with PC3 or PC3.PSMA cells. Results from day 12 are shown.

[0115] Figure 4A The results are shown using the switch receptor PD1*PTM.CD28 or PD1.CD28 linked to each individual PSMALenti CAR via F2A and transduced into primary human T cells. PD1 and CAR expression were examined by flow cytometry on day 12.

[0116] Figure 4B The results are shown using a dominant negative (dn) transforming growth factor beta receptor II (TGFRβII) sequence linked to each human PSMA Lenti CAR via T2A. At day 7, T cells transduced with the Dn-TGFRβII-PSMA CAR were analyzed by flow cytometry.

[0117] Figure 4C Graphed are the results of electroporating various amounts of purified full-length PDL1 RNA into PC3.PSMA cells and examining PDL1 expression by flow cytometry on day 13.

[0118] Figure 4D Results are shown for PC3.PSMA cells electroporated with various PSMA Lenti CARs and subjected to CD107a assays. Cells were gated by CD3. Results from day 14 are shown.

[0119] Figure 4E Results are shown for PC3.PSMA cells electroporated with various PSMA Lenti CARs and subjected to CD107a assays. Cells were gated by CD3. Results from day 14 are shown.

[0120] Figure 4F Results are shown for PC3.PSMA cells electroporated with various PSMA Lenti CARs and subjected to CD107a assays. Cells were gated by CD3. Results from day 14 are shown.

[0121] Figure 4G Results are shown for PC3.PSMA cells electroporated with various PSMA Lenti CARs and subjected to CD107a assays. Cells were gated by CD3. Results from day 14 are shown.

[0122] Figure 4H Results are shown for a luciferase-based CTL assay using various PSMA Lenti CARs cultured with PC3.PSMA cells. Results are reported as percent killing based on luciferase activity in wells with tumor alone in the absence of T cells.

[0123] Figure 4I Shown are the results of ELISA assays performed using various PSMA Lenti CARs incubated with PC3.PSMA or PDL1 electroporated PC3.PSMA cells (IL-2, upper panel; IFN-γ, lower panel).

[0124] Figure 5A Results are shown for primary human T cells transduced with the switch receptor PD1.CD28 linked to each human PSMA Lenti CAR via F2A. PD1 and CAR expression were examined by flow cytometry.

[0125] 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.

[0126] Figure 5C Results are shown for PC3.PSMA.7SC cells cultured with various PSMA Lenti CARs and subjected to a CD107a assay. Cells were gated by CD3.

[0127] Figure 5D Results are shown for PC3.PSMA.7SC cells electroporated with PDL1 using various PSMA Lenti CARs and subjected to a CD107a assay. Cells were gated by CD3.

[0128] Figure 5E Results are shown for a luciferase-based CTL assay using various PSMA Lenti CARs co-cultured with tumor cells. Results are reported as percent killing based on luciferase activity in wells with tumor alone in the absence of T cells.

[0129] Figure 5FShown are the results of ELISA assays performed on PC3, PC3.PSMA.7SC, or PDL1 electroporated PC3.PSMA.PDL1 cells incubated with various PSMA Lenti CARs (IL-2, upper panel; IFN-γ, lower panel).

[0130] Figure 5G Results of PSMA expression using quantitative PCR are shown. Fold changes (ΔΔCT) were normalized to Nalm6.CBG cells. For abbreviations, see Table 1.

[0131] Figure 5H Results are shown for various PSMA Lenti CARs cultured with tumor cells or primary human cells and assayed for CD107a. Cells were gated by CD3.

[0132] Figure 5I Shows Figure 5H Quantitative data from the experiments shown in. HSAEpC: human small airway epithelial cells. HPMEC: human pulmonary microvascular endothelial cells.

[0133] Figure 5J Results are shown for ELISA assays using various PSMA LentiCARs co-cultured with primary human cells (IL-2, left panel; IFN-γ, right panel). HREpC: human renal epithelial cells. HSAEpC: human small airway epithelial cells. HPMEC: human pulmonary microvascular endothelial cells.

[0134] Figure 5K Shown is the use of 2x10 6 After 27 days, 2x10 6 PSMA CAR-T positive transduced T cells were injected into tumor-bearing mice (iv). Bioluminescence imaging (BLI) was performed at multiple time points. The minimum average radiance of the above image is 5x 10 5 ; The minimum average radiance of the image below is 3 x 10 5 .

[0135] Figure 5L Illustrated Figure 5K The quantitative average radiance.

[0136] Figure 6 is a schematic representation of the dn-TGFRβII PSMA CAR construct and the pTRPE construct.

[0137] Figure 7Shown are flow cytometric examinations of CAR expression in T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS) or co-transduced with 2F5 PSMA CAR along with various switch receptors, as indicated.

[0138] Figure 8 Shown are flow cytometric examinations of PD1 and Tim3 expression in T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS) or co-transduced with 2F5 PSMA CAR along with various switch receptors, as indicated.

[0139] Figure 9 Figure 2 is a graph depicting CD107a expression in T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or 2F5 PSMA CAR co-transduced with various switch receptors, as indicated. UTD means untransduced.

[0140] Figure 10 Figure 2 is a graph depicting granzyme B expression in T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or co-transduced with 2F5 PSMA CAR along with various switch receptors, as indicated. UTD means untransduced.

[0141] Figure 11A Figure 2 is a graph depicting IL-2 secretion by T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or 2F5 PSMA CAR co-transduced with various switch receptors, as indicated. NTD means not transduced.

[0142] Figure 11B Figure 2 is a graph depicting IFNγ secretion by T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or co-transduced with 2F5 PSMA CAR along with various switch receptors, as indicated. UTD means untransduced.

[0143] Figure 12A Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0144] Figure 12B Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0145] Figure 13 is a graph depicting tumor size in NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0146] Figure 14A Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0147] Figure 14B Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0148] Figure 14C Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0149] Figure 14D Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0150] Figure 14E Graphs depicting quantification of bioluminescence obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0151] Figure 14F Graphs depicting quantification of bioluminescence (left) and tumor size (right) obtained from imaging of NSG mice bearing PC3-PSMA.CBG-induced tumors treated with T cells transduced as indicated.

[0152] Figure 14G This table lists T cells transduced according to the indicated conditions in descending order of tumor control ability. YMNM Better than WT ICOS. PD1*BB was better than PD1*CD28 when it had ICOSz or ICOSzYMNM.

[0153] Figure 15A Graph showing that CART-PSMA-TGFβRdn cells (dnTGFBR2-T2A-Pbbz) demonstrated enhanced antigen-specific proliferation compared to CART-PSMA (Pbbz) during 42 days of co-culture and after repeated stimulation with PSMA-expressing tumor cells (arrows). CD19-BBz CART (19bbz) and transduced T cells (mock) were used as controls.

[0154] 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 untransduced cells as a control group (mock).

[0155] Figure 15C are photographs showing the location and systemic burden of tumors assessed weekly by bioluminescence imaging (BLI).

[0156] Figure 16 Diagram illustrating the study protocol used in the Phase 1 clinical trial.

[0157] Figure 17 Graph showing CAR-T cell kinetics assessed by qPCR of CART-PSMA-TGFβRdn DNA in subjects 32816-02, -04, and -05 in Cohort 1, and subjects 32816-06, -07, and -08 in Cohort 2.

[0158] Figure 18A is a graph showing significant increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) and ferritin, which were associated with a Grade 3 cytokine release syndrome event in subject 32816-06.

[0159] Figure 18B The figure shows a significant increase in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) and ferritin, which was associated with a grade 3 cytokine release syndrome event in subject 32816-07.

[0160] Figure 19 is a graph showing prostate-specific antigen (PSA) responses in Group 1 and Group 2 patients.

[0161] Figure 20A is a graph showing the expression of PSMA-TGFβRDN CART in subject 32816-07 (left y-axis, number of copies of genomic DNA / ug) and the level of IL-6 (right y-axis, pg / ml), indicating the cytokine release syndrome that developed in the subject one day after infusion.

[0162] Figure 20B is a graph showing that management of cytokine release syndrome is accompanied by transient PSA reduction, as measured by C-reactive protein (CRP; left y-axis, mg / L) levels and serum ferritin levels (right y-axis, ng / L).

[0163] Figure 21is a graph showing the number of PSMA-positive circulating tumor cells (CTCs) detected in each subject over time. DETAILED DESCRIPTION

[0164] The present invention provides compositions and methods of modified immune cells, such as T cells and NK cells, or their precursor cells, such as modified T cells, which include chimeric antigen receptors (CAR). In some embodiments, CAR includes 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 modified immune cells include PSMA-CAR, which includes a murine PSMA binding domain. In some embodiments, the modified immune cells include PSMA-CAR, which includes a human PSMA binding domain. Methods for producing such genetically engineered cells are also provided. In some embodiments, cells and compositions can be used for adoptive cell therapy, such as adoptive tumor immunotherapy.

[0165] In some embodiments, the immune cells provided include additional receptors, such as dominant negative receptors and / or conversion receptors, so as to enhance the efficacy of immune cells in the tumor microenvironment. Such cells can change or reduce the effect of immunosuppressive signals in the tumor microenvironment. The modified immune cells of the present invention offset the upregulation and / or expression of inhibitory receptors or ligands that can negatively control T cell activation and T cell 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 effectiveness and efficacy of adoptive T cell therapy. For example, the expression of TGF-β on T cells and / or in the tumor microenvironment can reduce the effectiveness and efficacy of adoptive T cell therapy. In the context of adoptive cell therapy, such immunosuppressive signals may otherwise damage certain desired effector functions. Tumor cells and / or cells in the tumor microenvironment typically upregulate immunosuppressive proteins, such as PD-L1, to deliver immunosuppressive signals. Such immunosuppressive proteins can also be upregulated on T cells in the tumor microenvironment, for example, on tumor-infiltrating T cells, which can occur after signaling through antigen receptors or certain other activation signals. Such events can contribute to the exhaustion phenotype of genetically engineered immune cells (e.g., targeting PSMA) T cells, which can in turn lead to reduced function when present in the vicinity of other cells expressing such proteins. Thus, the modified immune cells of the present invention address T cell exhaustion and / or lack of T cell persistence, which are obstacles to the efficacy and therapeutic outcomes of conventional adoptive cell therapy.

[0166] The present invention includes PSMA CAR and its use in treating cancer. In certain embodiments, the present invention includes human PSMA CAR with dominant negative receptors and / or conversion receptors. One of the major obstacles to cancer immunotherapy is the tumor microenvironment. Upregulation of immunosuppressive molecules (such as PD-1) negatively regulates T cell activity.

[0167] The present invention is based on the discovery that T cells comprising PSMA-CAR and dominant negative and / or switch receptors are able to bypass the effects of immunosuppressive molecules in the tumor microenvironment, providing continuous and potent anti-tumor activity.

[0168] It should be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions may vary. It should also be understood that the terminology used herein is only used to describe specific embodiments and is not intended to limit the present invention.

[0169] In addition, unless otherwise stated, the experiments described herein use conventional molecular and cell biology and immunological techniques within the scope of the art. Such techniques are known to those skilled in the art and have been fully explained in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (4th edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0170] A. Definition

[0171] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which the invention pertains. If there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Unless otherwise indicated, the use of "or" means "and / or". The term "including" and other forms of terms such as "includes" and "included" are non-restrictive.

[0172] Generally, the nomenclature used in conjunction with cell and tissue culture as herein described, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization is well known in the art and commonly used. Method and technology provided herein are usually carried out according to conventional methods known in the art, and as described in the various general and more specific references quoted and discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as generally realized in this area or as described herein. The terms and laboratory procedures and techniques relevant to analytical chemistry as herein described, synthetic organic chemistry and drugs and medicinal chemistry are well known in the art and commonly used. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery and patient's treatment.

[0173] The following definitions of selected terms may make the present disclosure more easily understood.

[0174] The articles "a" and "an" are used herein to refer to one or to more than one (ie, 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.

[0175] As used herein, "about," when referring to a measurable value such as an amount, a time period, and the like, is meant to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the given value, as long as such variations are appropriate to practice the disclosed methods.

[0176] "Activation," as used herein, refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" and the like refer to a T cell that has undergone cell division.

[0177] As used herein, the term "alleviating" a disease means reducing the severity of one or more symptoms of a disease.

[0178] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, and can be immunoreactive portions of intact immunoglobulins (e.g., binding fragments of an antibody). Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present invention can exist in a variety of 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).

[0179] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multispecific antibodies.

[0180] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in their naturally occurring conformation in all antibody molecules.

[0181] "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in their naturally occurring conformations in all antibody molecules, alpha and beta light chains referring to the two major antibody light chain isotypes.

[0182] The term "synthetic antibody," as used herein, refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to refer to an antibody that has been produced by the synthesis of a DNA molecule encoding the antibody and expressing the antibody protein or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology available and widely known in the art.

[0183] As used herein, the term "antigen" or "Ag" is defined as a molecule that stimulates an immune response. The immune response may involve the production of antibodies, or the activation of specific immune competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0184] In addition, antigens can be derived from recombinant or genomic DNA. It will be understood by those skilled in the art that any DNA, including nucleotide sequences or partial nucleotide sequences encoding proteins that cause an immune response, encodes the term "antigen" as used herein. In addition, it will be understood by those skilled in the art that antigens need not be encoded solely by the full-length nucleotide sequence of a gene. It will be 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 different combinations to cause a desired immune response. Moreover, it will be understood by those skilled in the art that antigens do not necessarily have to be encoded by a "gene" at all. It will be readily apparent that antigens can be produced, synthesized, or can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0185] As used herein, the term "autologous" refers to any substance originating from the same individual that is subsequently reintroduced into that individual. "Allogeneic" refers to any substance originating from a different animal of the same species. "Xenogeneic" refers to any substance originating from an animal of a different species.

[0186] 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 binds to an antigen. CAR can be used as adoptive cell transfer therapy. T cells are removed from the patient's body and modified to express receptors that are specific for the antigen or a specific form of the antigen. In some embodiments, CAR is specific for a selected target, such as cells that express prostate-specific membrane antigen. CAR can also include an intracellular activation domain, a transmembrane domain, and an extracellular domain, the extracellular domain including a tumor-associated antigen binding region.

[0187] As used herein, the term "costimulatory ligand" includes molecules on antigen presenting cells (e.g., artificial APCs (aAPCs), dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on T cells, 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, for example, binding of the TCR / CD3 complex to an MHC molecule loaded with a peptide. Costimulatory ligands may 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. Co-stimulatory ligands also include, in particular, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphoid function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0188] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of 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.

[0189] As used herein, "co-stimulatory signal" refers to a signal that, in conjunction with a primary signal (such as TCR / CD3 ligation), results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0190] A "disease" is a health state in an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be if it were not in the disorder. Remaining untreated, the disorder does not necessarily cause a further deterioration in the animal's health state.

[0191] As used herein, the term "downregulate" refers to reducing or eliminating gene expression of one or more genes.

[0192] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition as described herein that is effective to achieve a specific biological result, or to provide a therapeutic or preventive benefit. Such results may include, but are not limited to, an amount of the composition that, when administered to a mammal, causes a detectable level of immunosuppression or immune tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed using a number of methods recognized in the art. It will be understood by those skilled in the art that the amount of the composition administered herein varies and can be readily determined based on many 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 specific compound being administered.

[0193] The term "coding" refers to the intrinsic properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) as a template for synthesizing other polymers and macromolecules with a definite nucleotide sequence (e.g., rRNA, tRNA and mRNA) or definite amino acid sequence in a biological process, and the resulting biological properties. Therefore, if transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes the protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and non-coding strand (as a template for transcription of a gene or cDNA) can be referred to as coded protein or other products of the gene or cDNA.

[0194] As used herein, the term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0195] As used herein, the term "epitope" is defined as a small chemical molecule on an antigen that can trigger an immune response, inducing a B and / or T cell response. An antigen may have one or more epitopes. Most antigens have multiple epitopes; that is, these antigens are multivalent. Typically, the size of an epitope is about 10 amino acids and / or sugars. Preferably, an epitope is about 4 to 18 amino acids, more preferably about 5 to 16 amino acids, and even more preferably 6 to 14 amino acids, more preferably about 7 to 12 amino acids, and most preferably about 8 to 10 amino acids. It will be understood by those skilled in the art that, in general, the main condition for antigen specificity is the overall three-dimensional structure of the molecule, rather than its specific linear sequence, thereby distinguishing different epitopes. Based on the present disclosure, the peptides of the present invention can be epitopes.

[0196] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0197] 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, the number of T cells expanded ex vivo is increased relative to the number initially present in the culture medium. In another embodiment, the number of T cells expanded ex vivo is increased relative to other cell types in the culture medium. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (such as a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0198] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0199] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may 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) that incorporate a recombinant polynucleotide.

[0200] " humanized " form of non-human (such as murine) antibody is chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab ', F (ab ') 2 or other antigen binding subsequences of antibody), which contains the minimum sequence from non-human immunoglobulin. In most cases, humanized antibody is human immunoglobulin (receptor antibody), wherein the residues from the complementary determining region (CDR) of receptor are replaced by the residues of the CDR with required specificity, affinity and capacity from non-human species (such as mouse, rat or rabbit) (donor antibody). In some cases, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibody can include residues that are neither found in the CDR or framework sequence of receptor antibody nor in the imported one. These modifications can further improve and optimize antibody performance. Generally, a humanized antibody will include substantially all of the following: at least one and typically two variable domains, wherein 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 those of a human immunoglobulin sequence. A humanized antibody may also include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. 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 its binding fragment, in which the entire molecule is of human origin or consists of an amino acid sequence identical to that of the human form of the antibody.

[0201] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that act as antibodies. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response of most subjects. It is the most effective immunoglobulin in agglutination reactions, complement fixation, and other antibody responses, and is important in protecting against bacteria and viruses. IgD is an immunoglobulin that does not have a known antibody function but can be used as an antigen receptor. IgE is an immunoglobulin that mediates immediate allergic reactions after exposure to allergens by causing the release of mediators from mast cells and basophils.

[0202] As used herein, the term "identity" refers to the subunit sequence identity between two polymeric molecules, particularly between two amino acid molecules, such as two polypeptide molecules. When the same position of two amino acid sequences has the same residue, for example, if a position in each of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue in the same aligned position is usually expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions, for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 amino acids in length) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0203] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes recognize the antigenic molecule as foreign and induce antibody formation and / or activate lymphocytes to remove the antigen.

[0204] As used herein, the term "immunosuppression" refers to a decrease in the overall immune response.

[0205] The term "isolated" means altered or removed from its native state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its native 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).

[0206] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most effective gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide tools for achieving significant levels of gene transfer in vivo.

[0207] As used herein, the term "modified" refers to an alteration in the state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0208] 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 the treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. The term includes perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0209] In the context of the present invention, the following abbreviations are used for commonly occurring nucleic acid bases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0210] 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" may also include introns, to the extent that a nucleotide sequence encoding the protein may include intron(s) in certain versions.

[0211] The term "operably linked" or "operatively linked" refers to a functional connection 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 to 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.

[0212] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. In addition, nucleic acid is a polymer of nucleotides. Therefore, nucleic acid and polynucleotide as used herein are interchangeable. It is generally known to those skilled in the art that nucleic acids are polynucleotides, which 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 and by synthetic means, any means including, but not limited to, recombinant means, i.e., cloning from a recombinant library or a cell genome nucleic acid sequence using conventional cloning techniques and PCR, etc.

[0213] 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 constitute a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, such as those commonly referred to in the art as peptides, oligopeptides, and oligomers, and also to longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0214] As used herein, the term "specific binding" 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 the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, this cross-species reactivity itself does not change the specificity of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the specificity of the antibody. In some examples, the term "specific binding" or "specifically binds" can be used with reference to the interaction between an antibody, protein, or peptide and a second chemical species to refer to 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 generally recognizing and binding proteins. If the antibody is specific for epitope "A," the presence of molecules comprising epitope A (or free, unlabeled A) in a reaction comprising labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0215] The term "stimulation" refers to a primary response induced by 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 via the TCR / CD3 complex). Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structure.

[0216] "Stimulatory molecule," as the term is used herein, refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0217] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B-cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response of 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, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0218] The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, canines, felines, and murine mammals. Preferably, the subject is a human.

[0219] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of the nucleic acid to which a binding molecule will specifically bind under conditions sufficient for binding to occur.

[0220] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is achieved by the inhibition, alleviation or eradication of the disease state.

[0221] "Graft" refers to the biocompatible lattice or donor tissue, organ, or cells to be transplanted. Examples of transplants include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Graft may also refer to any material to be administered to a host. For example, a graft may refer to a nucleic acid or protein.

[0222] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0223] As used herein, the term "treat," "treat," or "treating" refers to reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0224] A "vector" is a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmids 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, retroviral vectors, lentiviral vectors, etc.

[0225] Range: Throughout this disclosure, various aspects of the invention may be presented in the form of ranges. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the invention. Thus, descriptions of ranges should be considered to specifically disclose all possible subranges as well as single numbers within the range. For example, description of a range from 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 single numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the size of the range.

[0226] B. Chimeric Antigen Receptor

[0227] The present invention provides compositions and methods of modified immune cells or their precursors (such as modified T cells), which include chimeric antigen receptors (CARs). Therefore, in some embodiments, immune cells have been genetically modified to express CARs. The CARs of the present invention include an antigen binding domain, a transmembrane domain, a hinge domain, and an intracellular signaling domain.

[0228] The antigen binding domain can be operably connected to another domain of CAR, such as a transmembrane domain or an intracellular domain described elsewhere herein, for expression in a cell. In one embodiment, the first nucleic acid sequence encoding the antigen binding domain is operably connected to the second nucleic acid encoding the transmembrane domain, and is further operably connected to a third nucleic acid sequence encoding the intracellular domain.

[0229] The antigen binding domains described herein can be combined with any transmembrane domain described herein, any intracellular domain or cytoplasmic domain described herein, or any other domain described herein that can be included in the CAR of the present invention. The subject CAR of the present invention may also include a spacer domain as described herein. In some embodiments, the antigen binding domain, the transmembrane domain, and the intracellular domain are separated by a linker.

[0230] Antigen binding domain

[0231] The antigen binding domain of CAR is the extracellular region of CAR for binding to specific target antigens, including proteins, carbohydrates and glycolipids. In some embodiments, CAR includes affinity for target antigens on target cells. Target antigens may include any type of protein or epitope thereof associated with target cells. For example, CAR may include affinity for target antigens on target cells, which indicates a specific disease state of the target cell.

[0232] In an exemplary embodiment, the target cell antigen is prostate-specific membrane antigen (PSMA). PSMA is a membrane-bound protein expressed on the cell surface and is reported to be highly overexpressed in prostate cancer tissue. PSMA expression is directly correlated with progression tumor grade and stage and is believed to confer a selective growth advantage on prostate cancer cells. Thus, the exemplary CAR of the present disclosure is specific for PSMA on target cells.

[0233] As described herein, the CARs of the present disclosure that have affinity for a specific target antigen on a target cell can include a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target-specific binding domain is of murine 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 exemplary embodiments, the CARs of the present disclosure that have affinity for PSMA on a target cell can include a PSMA binding domain. In some embodiments, the PSMA binding domain is a murine PSMA binding domain, e.g., the PSMA binding domain is of murine origin. In some embodiments, the PSMA binding domain is a human PSMA binding domain, e.g., the PSMA binding domain is of human origin.

[0234] 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, CAR may have affinity for one or more target antigens on target cells. In such embodiments, CAR is a bispecific CAR, or a multispecific CAR. In some embodiments, CAR includes one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, CAR includes 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 bind to different epitopes of the target antigen. When multiple target-specific binding domains are present in CAR, the binding domains can be arranged in series and can be 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 on a single polypeptide chain through an oligopeptide or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0235] In some embodiments, the antigen binding domain is selected from 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 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.

[0236] The antigen binding domain may include any domain that binds to an antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antigen, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. In some embodiments, the antigen binding domain portion includes a mammalian antibody or a fragment thereof. The selection of the antigen binding domain may depend on the type and number of antigens present on the surface of the target cell.

[0237] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked via an encoded peptide linker that connects the N-terminus of the VH to the C-terminus of the VL, or vice versa. In some embodiments, the antigen-binding domain (e.g., a PSMA-binding domain) comprises an scFv with a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain (e.g., a PSMA-binding domain) comprises an scFv with a VL-linker-VH configuration from the N-terminus to the C-terminus. One skilled in the art will be able to select an appropriate configuration for use in the present invention.

[0238] The linker is typically 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 WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including but not limited to glycine serine (GS) linkers, such as (GS) n 、(GSGGS) n (SEQ ID NO: 1), (GGGS) n(SEQ ID NO: 2) and (GGGGS) n (SEQ ID NO: 3), wherein n represents an integer of at least 1. Exemplary linker sequences may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 4), GGSGG (SEQ ID NO: 5), GSGSG (SEQ ID NO: 6), GSGGG (SEQ ID NO: 7), GGGSG (SEQ ID NO: 8), GSSSG (SEQ ID NO: 9), GGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), etc. Those skilled in the art will be able to select suitable linker sequences for use in the present invention. In one embodiment, an antigen binding domain (e.g., a PSMA binding domain) of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 11), which can be encoded by the nucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO: 12).

[0239] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids comprising 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 Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, 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 25 278(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).

[0240] As used herein, "Fab" refers to a fragment of an antibody structure that binds antigen but is monovalent and does not have an Fc portion, e.g., papain digestion of an antibody produces two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; the Fc region that does not bind antigen).

[0241] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of an intact IgG antibody, wherein the fragment has two antigen-binding (ab') (divalent) regions, wherein each (ab') region comprises two independent amino acid chains, a portion of the H chain and the light (L) chain linked by an SS bond for antigen binding, and wherein the remaining H chains are linked together. The "F(ab')2" fragment can be divided into two independent Fab' fragments.

[0242] In some embodiments, the antigen binding domain can be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR can include a human antibody described elsewhere herein, or a fragment thereof.

[0243] In an exemplary embodiment, the PSMA-CAR of the present invention comprises a PSMA-binding domain, e.g., a PSMA-specific scFv.

[0244] (a) Murine PSMA-binding domain and its variants.

[0245] In certain embodiments, the PSMA-CAR of the present invention comprises a murine PSMA-binding domain or a variant thereof.

[0246] In certain embodiments, the PSMA-CAR of the present invention includes a PSMA binding domain of a non-human PSMA antibody (e.g., a mouse or rat PSMA antibody), or a variant thereof. It is well known in the art that murine or other non-human antibodies can be raised by immunizing a non-human (e.g., mouse) with human PSMA or a fragment thereof.

[0247] In one embodiment, the PSMA binding domain is the murine J591 PSMA binding domain, which is comprised within the amino acid sequence set forth below:

[0248] MALPPVTALLLPLALLLHAARPGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGG GGSGGGGSSGGGSEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:14),

[0249] It can be encoded by the nucleic acid sequence described below:

[0250] ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC(SEQ ID NO:15)。

[0251] Those skilled in the art are aware of the permissible variations of the murine J591 PSMA-binding domain while maintaining binding to PSMA. For example, in some embodiments, the PSMA-binding domain is a murine 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 murine J591 PSMA-binding domain amino acid sequence comprised in SEQ ID NO: 14. In one embodiment, the PSMA binding domain is the murine J591 PSMA binding domain encompassed by the amino acid sequence set forth in SEQ ID NO:14.

[0252] 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%, at least 99% sequence identity to the murine J591 PSMA binding domain coding sequence 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 recited in SEQ ID NO: 15.

[0253] In an exemplary embodiment, the PSMA-CAR of the present invention comprises a PSMA binding domain, such as a PSMA-specific scFv. In one embodiment, the PSMA binding domain is a murine J591 PSMA binding domain comprising the amino acid sequence described below:

[0254] DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGG GSEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:13),

[0255] It can be encoded by the nucleic acid sequence described below:

[0256] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC(SEQ ID NO:180)。

[0257] Those skilled in the art will appreciate the permissible variations of the murine J591 PSMA-binding domain while maintaining binding to human PSMA. For example, in some embodiments, the PSMA-binding domain is a murine 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 murine J591 PSMA-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0258] 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.

[0259] In one embodiment, the murine J591 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

[0260] DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVP DRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK(SEQ ID NO:16),

[0261] It can be encoded by the nucleic acid sequence described below:

[0262] GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGG CACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTTCCACGTTCGGTGCTGGGACCATGCTGGACCTGAAA(SEQ ID NO:17).

[0263] Those skilled in the art will appreciate the permissible variations in the light chain variable region while maintaining its contribution to human RPSMA binding. 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.

[0264] 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%, 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.

[0265] In one embodiment, the murine J591 PSMA binding domain comprises the light chain variable region described in NCBI GenBank sequence database ID: CCA78125.1, which comprises the amino acid sequence set forth below:

[0266] DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLAITNVQSEDLADYFCQQYNSYPLTFGAGTKLEIKR (SEQ ID NO: 181).

[0267] Those skilled in the art will appreciate the permissible variations in the light chain variable region while maintaining its contribution to human PSMA binding. 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%, 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 murine J591 PSMA-binding domain includes 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 murine J591 PSMA-binding domain can include a light chain variable region comprising a CDR1 represented by the amino acid sequence KASQDVGTAVD (SEQ ID NO: 18); a CDR2 represented by the amino acid sequence WASTRHT (SEQ ID NO: 19); and a CDR3 represented by the amino acid sequence QQYNSYPLT (SEQ ID NO: 20). One skilled in the art will appreciate the permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding. For example, the murine J591 PSMA-binding domain can include a light chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the murine J591 PSMA-binding domain can include a light chain variable region comprising a CDR2 comprising an amino acid sequence that has 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 murine J591 PSMA-binding domain can include 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 foregoing light chain variable region CDRs.

[0268] In one embodiment, the murine J591 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth below:

[0269] EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:21),

[0270] It can be encoded by the nucleic acid sequence described below:

[0271] GAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACC ACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC(SEQ ID NO:22).

[0272] Those skilled in the art will appreciate the permissible variations in the heavy chain variable region while maintaining its contribution to human PSMA binding. 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.

[0273] 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.

[0274] In one embodiment, the murine J591 PSMA binding domain comprises the heavy chain variable region described in NCBI GenBank sequence database ID: CCA78124.1, which comprises the amino acid sequence set forth below:

[0275] EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS (SEQ ID NO: 182).

[0276] Those skilled in the art will appreciate the permissible variations in the heavy chain variable region while maintaining its contribution to PSMA binding. 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.

[0277] The heavy chain variable region of the murine J591 PSMA-binding domain includes three heavy chain complementarity-determining regions (CDRs). Thus, the murine J591 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 represented by the amino acid sequence GYTFTEYTIH (SEQ ID NO: 23); a CDR2 represented by the amino acid sequence NINPNNNGGTTYNQKFED (SEQ ID NO: 24); and a CDR3 represented by the amino acid sequence GWNFDY (SEQ ID NO: 25). One skilled in the art will appreciate the permissible variations in the heavy chain CDRs while maintaining their contribution to binding to human PSMA. For example, the murine J591 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the murine J591 PSMA-binding domain can include a heavy chain variable region comprising a CDR2 comprising an amino acid sequence that has 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 murine J591 PSMA-binding domain can include 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 foregoing heavy chain variable region CDRs.

[0278] In one embodiment, the PSMA binding domain is a murine J591 PSMA binding domain comprising an amino acid sequence comprising 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 sequences set forth in SEQ ID NOs: 16 and 21.

[0279] In one embodiment, the PSMA binding domain is a murine J591 PSMA binding domain comprising an amino acid sequence comprising 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 NOs: 181 and 182.

[0280] (b) Humanized PSMA-binding domain

[0281] 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 variant or fragment thereof. In certain exemplary embodiments, the PSMA CAR comprises a humanized variant of the murine J591 antibody that binds to human PSMA. Methods for humanizing murine antibodies are known in the art.

[0282] In one embodiment, the PSMA-binding domain is a humanized PSMA-specific binding domain. In certain embodiments, the PSMA-binding domain is a humanized J591 PSMA-binding domain. In certain embodiments, the PSMA-binding domain comprises any of the heavy 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 may comprise an scFv comprising any of the heavy and light chain variable regions disclosed therein. Thus, the PSMA-CAR of the present invention comprises a humanized variant of the murine J591 antibody that binds to human PSMA disclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1.

[0283] In certain embodiments, a PSMA-binding domain of the invention may comprise a heavy chain variable region and a light chain variable region of any of those recited in Table 19:

[0284] Table 19: Humanized PSMA-binding heavy and light chain variable sequences

[0285]

[0286]

[0287]

[0288] (c) Human PSMA-binding domain

[0289] 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 is a human 1C3 PSMA binding domain comprising the amino acid sequence described below:

[0290] MALPPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVW GQGTTVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK(SEQ ID NO:26),

[0291] It can be encoded by the nucleic acid sequence described below:

[0292] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:27)。

[0293] Those skilled in the art will appreciate the permissible variations of human 1C3 PSMA while maintaining binding to human PSMA. 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.

[0294] 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.

[0295] In one embodiment, the human 1C3 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence recited below:

[0296] PQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 28),

[0297] It can be encoded by the nucleic acid sequence described below:

[0298] CCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGAC TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:29).

[0299] Those skilled in the art will appreciate the permissible variations in the heavy chain variable region while maintaining its contribution to human PSMA binding. 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.

[0300] 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.

[0301] The heavy chain variable region of the human 1C3 PSMA-binding domain includes three heavy chain complementarity-determining regions (CDRs). Thus, the human 1C3 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 represented by the amino acid sequence SYAMH (SEQ ID NO: 30); a CDR2 represented by the amino acid sequence VISYDGNNKYYADSVKG (SEQ ID NO: 31); and a CDR3 represented by the amino acid sequence AVPWGSRYYYYGMDV (SEQ ID NO: 32). One skilled in the art will appreciate the permissible variations in the heavy chain CDRs while maintaining their contribution to PSMA binding. For example, the human 1C3 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 1C3 PSMA-binding domain can include 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 include 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 foregoing heavy chain variable region CDRs.

[0302] In one embodiment, the human 1C3 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence recited below:

[0303] AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK (SEQ ID NO: 33), which may be encoded by the nucleic acid sequence described below:

[0304] GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTG GAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:34).

[0305] Those skilled in the art will appreciate the permissible variations in the light chain variable region while maintaining its contribution to human PSMA binding. 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.

[0306] 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.

[0307] The light chain variable region of the human 1C3 PSMA-binding domain includes three light chain complementarity determining regions (CDRs). Thus, the human 1C3 PSMA-binding domain can include a light chain variable region comprising a CDR1 represented by the amino acid sequence RASQGISSALA (SEQ ID NO: 35); a CDR2 represented by the amino acid sequence DASSLES (SEQ ID NO: 36); and a CDR3 represented by the amino acid sequence QQFNSYPLT (SEQ ID NO: 37). One skilled in the art will appreciate the permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding. For example, the human 1C3 PSMA-binding domain can include a light chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 1C3 PSMA-binding domain can include 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 include 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 CDR1 amino acid sequence set forth in SEQ ID NO: 37. In one embodiment, a human 1C3 PSMA-binding domain comprises a light chain variable region comprising three of the aforementioned light chain variable region CDRs.

[0308] In one embodiment, the PSMA-binding domain is the human 2A10 PSMA-binding domain comprising the amino acid sequence recited below:

[0309] MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK(SEQ ID NO:38),

[0310] It can be encoded by the nucleic acid sequence described below:

[0311] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:39)。

[0312] Those skilled in the art will appreciate the permissible variations of human 2A10 PSMA while maintaining binding to human PSMA. 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.

[0313] 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.

[0314] In one embodiment, the human 2A10 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence recited below:

[0315] PEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSS (SEQ ID NO:40),

[0316] It can be encoded by the nucleic acid sequence described below:

[0317] CCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATA CAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA(SEQ ID NO:41).

[0318] Those skilled in the art will appreciate the permissible variations in the heavy chain variable region while maintaining its contribution to human PSMA binding. 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.

[0319] 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.

[0320] The heavy chain variable region of the human 2A10 PSMA-binding domain includes three heavy chain complementarity-determining regions (CDRs). Thus, the human 2A10 PSMA-binding domain may include a heavy chain variable region comprising a CDR1 represented by the amino acid sequence SNWIG (SEQ ID NO: 42); a CDR2 represented by the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 43); and a CDR3 represented by the amino acid sequence QTGFLWSSDL (SEQ ID NO: 44). One skilled in the art will appreciate the permissible variations in the heavy chain CDRs while maintaining their contribution to binding to human PSMA. For example, the human 2A10 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 2A10 PSMA-binding domain can include a heavy chain variable region comprising a CDR2 comprising an amino acid sequence that has 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, the human 2A10 PSMA-binding domain can include 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 includes a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

[0321] In one embodiment, the human 2A10 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

[0322] AIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK (SEQ ID NO: 45), which may be encoded by the nucleic acid sequence described below:

[0323] GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTG GAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:46).

[0324] Those skilled in the art will appreciate the permissible variations in the light chain variable region while maintaining its contribution to human PSMA binding. 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 binding domain comprising the amino acid sequence set forth in SEQ ID NO:45.

[0325] 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.

[0326] The light chain variable region of the human 2A10 PSMA-binding domain includes three light chain complementarity-determining regions (CDRs). Thus, the human 2A10 PSMA-binding domain can include a light chain variable region comprising a CDR1 represented by the amino acid sequence CRASQDISSAL (SEQ ID NO: 47); a CDR2 represented by the amino acid sequence YDASSLES (SEQ ID NO: 48); and a CDR3 represented by the amino acid sequence CQQFNSYPLT (SEQ ID NO: 49). One skilled in the art will appreciate the permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding. For example, the human 2A10 PSMA-binding domain can include a light chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 2A10 PSMA-binding domain can include 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, the human 2A10 PSMA-binding domain can include 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 includes a light chain variable region comprising three of the aforementioned light chain variable region CDRs.

[0327] In one embodiment, the PSMA-binding domain is a human 2F5 PSMA-binding domain comprising the amino acid sequence recited below:

[0328] MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGT LVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIK(SEQ ID NO:50),

[0329] It can be encoded by the nucleic acid sequence described below:

[0330] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAA(SEQ ID NO:51)。

[0331] Those skilled in the art will appreciate the permissible variations of human 2F5 PSMA while maintaining binding to human PSMA. 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.

[0332] 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.

[0333] In one embodiment, the human 2F5 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence recited below:

[0334] PEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSS (SEQ ID NO: 52),

[0335] It can be encoded by the nucleic acid sequence described below:

[0336] CCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATA CAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA(SEQ ID NO:53).

[0337] Those skilled in the art will appreciate the permissible variations in the heavy chain variable region while maintaining its contribution to human PSMA binding. 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.

[0338] 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.

[0339] The heavy chain variable region of the human 2F5 PSMA-binding domain includes three heavy chain complementarity-determining regions (CDRs). Thus, the human 2F5 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 represented by SNWIG (SEQ ID NO: 54); a CDR2 represented by the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 55); and a CDR3 represented by the amino acid sequence QTGFLWSFDL (SEQ ID NO: 56). One skilled in the art will appreciate the permissible variations in the heavy chain CDRs while maintaining their contribution to PSMA binding. For example, the human 2F5 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 2F5 PSMA-binding domain can include 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 include a heavy chain variable region comprising a CDR3 comprising an amino acid sequence that has 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, a human 2F5 PSMA-binding domain comprises a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

[0340] In one embodiment, the human 2F5 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence recited below:

[0341] AIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIK (SEQ ID NO: 57), which may be encoded by the nucleic acid sequence described below:

[0342] GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAA AGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAA(SEQ ID NO:58).

[0343] Those skilled in the art will appreciate the permissible variations in the light chain variable region while maintaining its contribution to human PSMA binding. 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%, 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.

[0344] 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.

[0345] The light chain variable region of the human 2F5 PSMA-binding domain includes three light chain complementarity determining regions (CDRs). Thus, the human 2F5 PSMA-binding domain can include a light chain variable region comprising a CDR1 represented by the amino acid sequence RASQDISSALA (SEQ ID NO: 59); a CDR2 represented by the amino acid sequence DASSLES (SEQ ID NO: 60); and a CDR3 represented by the amino acid sequence QQFNSYPLT (SEQ ID NO: 61). One skilled in the art will appreciate the permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding. For example, the human 2F5 PSMA-binding domain can include a light chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 2F5 PSMA-binding domain can include 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 include 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, a human 2F5 PSMA-binding domain comprises a light chain variable region comprising three of the aforementioned light chain variable region CDRs.

[0346] In one embodiment, the PSMA-binding domain is a human 2C6 PSMA-binding domain comprising the amino acid sequence recited below:

[0347] MALPVTALLLPLALLLHAARPEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQ GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLFTFGPGTKVDIK(SEQ ID NO:62),

[0348] It can be encoded by the nucleic acid sequence described below:

[0349] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA(SEQ ID NO:63)。

[0350] Those skilled in the art will appreciate the permissible variations of human 2C6 PSMA while maintaining binding to human PSMA. 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.

[0351] 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.

[0352] In one embodiment, the human 2C6 PSMA-binding domain comprises a heavy chain variable region comprising the amino acid sequence recited below:

[0353] PEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQGTLVTVSS(SEQ ID NO:64),

[0354] It can be encoded by the nucleic acid sequence described below:

[0355] CCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG CCCGTCCTTCCAAGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ IDNO:65).

[0356] Those skilled in the art will appreciate the permissible variations in the heavy chain variable region while maintaining its contribution to human PSMA binding. 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.

[0357] 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.

[0358] The heavy chain variable region of the human 2C6 PSMA-binding domain includes three heavy chain complementarity-determining regions (CDRs). Thus, the human 2C6 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 represented by the amino acid sequence TNYWI (SEQ ID NO: 66); a CDR2 represented by the amino acid sequence GIIYPGDSDTRYSPSFQG (SEQ ID NO: 67); and a CDR3 represented by the amino acid sequence SPGYTSSWTS (SEQ ID NO: 68). One skilled in the art will appreciate the permissible variations in the heavy chain CDRs while maintaining their contribution to PSMA binding. For example, the human 2C6 PSMA-binding domain can include a heavy chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 2C6 PSMA-binding domain can include 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 include 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, a human 2C6 PSMA-binding domain comprises a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

[0359] In one embodiment, the human 2C6 PSMA-binding domain comprises a light chain variable region comprising the amino acid sequence recited below:

[0360] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLFTFGPGTKVDIK (SEQ ID NO: 69), which may be encoded by the nucleic acid sequence described below:

[0361] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGC CACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA(SEQ ID NO:70).

[0362] Those skilled in the art will appreciate the permissible variations in the light chain variable region while maintaining its contribution to human PSMA binding. 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.

[0363] 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.

[0364] The light chain variable region of the human 2C6 PSMA-binding domain includes three light chain complementarity determining regions (CDRs). Thus, the human 2C6 PSMA-binding domain can include a light chain variable region comprising a CDR1 represented by the amino acid sequence CRASQSVSSYL (SEQ ID NO: 71); a CDR2 represented by the amino acid sequence YDASNRAT (SEQ ID NO: 72); and a CDR3 represented by the amino acid sequence CQQRSNWPLFT (SEQ ID NO: 73). One skilled in the art will appreciate the permissible variations in the light chain CDRs while maintaining their contribution to PSMA binding. For example, the human 2C6 PSMA-binding domain can include a light chain variable region comprising a CDR1 comprising an amino acid sequence that has 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, the human 2C6 PSMA-binding domain can include 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 include 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 aforementioned light chain variable region CDRs.

[0365] transmembrane domain

[0366] The CAR of the present invention (e.g., PSMA-CAR) can include a transmembrane domain that connects the antigen binding domain of CAR to the intracellular domain of CAR. The transmembrane domain of subject CAR is a region of the plasma membrane that can span a cell (e.g., an immune cell or its precursor). The transmembrane domain is used to insert into a cell membrane, such as a eukaryotic cell membrane. In some embodiments, the transmembrane domain is between the antigen binding domain and the intracellular domain of CAR.

[0367] In some embodiments, the transmembrane domain is naturally associated with one or more 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 to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0368] Transmembrane domain can be derived from natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein, such as, type I transmembrane protein.When the source is synthetic, the transmembrane domain can be any artificial sequence that promotes CAR to insert into the cell membrane, such as, artificial hydrophobic sequence. In the present invention, the example of the transmembrane domain of specific use includes but is not limited to the following transmembrane domain (that is, at least including the following transmembrane region (one or more)): α, β or ζ chain of T cell receptor, CD28, CD3ε, 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 comprise primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0369] The transmembrane domains described herein can be combined with any antigen binding domain described herein, any intracellular domain, or any other domain described herein that can be included in a subject CAR.

[0370] In some embodiments, the transmembrane domain further includes a hinge region. The subject CAR of the present invention may also include a hinge region. The hinge region of CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, the domain promotes the suitable protein folding of CAR. The hinge region is an optional component for CAR. The hinge region may include a domain selected from the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, the CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, CD8a hinges, artificial hinges prepared from polypeptides that can be as small as three glycine (Gly), and CH1 and CH3 domains of IgG (such as human IgG4).

[0371] In some embodiments, the subject CAR of the present disclosure includes a hinge region, which connects the antigen binding domain to the transmembrane domain, and then connects to the intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to identify and bind to the 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, thus allowing the antigen binding domain to have the specific structure and density of the target antigen on the optimal recognition cell (such as a tumor cell) (Hudecek et al., the same). The flexibility of the hinge region allows the hinge region to adopt many different conformations.

[0372] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (eg, a hinge region derived from CD8).

[0373] The hinge region can have a length of about 4 amino acids to about 50 amino acids, e.g., 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.

[0374] Suitable hinge regions can be readily selected and can have any number of suitable lengths, such as 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.

[0375] For example, the hinge region includes glycine polymers (G)n, glycine-serine polymers (including, for example, (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 unstructured and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine even enters significantly more than alanine. The hinge region can be spaced apart and less constrained than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 4), GGSGG (SEQ ID NO: 5), GSGSG (SEQ ID NO: 6), GSGGG (SEQ ID NO: 7), GGGSG (SEQ ID NO: 8), GSSSG (SEQ ID NO: 9), etc.

[0376] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., 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 non-limiting examples, the immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 74); CPPC (SEQ ID NO: 75); CPEPKSCDTPPPCPR (SEQ ID NO: 76) (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280: 41494-41503); ELKTPLGDTTHT (SEQ ID NO: 77); KSCDKTHTCP (SEQ ID NO: 78); KCCVDCP (SEQ ID NO: 79); KYGPPCP (SEQ ID NO: 80); EPKSCDKTHTCPPCP (SEQ ID NO: 81) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 82) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 83) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 84). NO:84) (human IgG4 hinge); etc.

[0377] The hinge region may comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. In one embodiment, the hinge region may 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 may be substituted with Tyr so that the hinge region comprises 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 may comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0378] The transmembrane domain can be combined with any hinge region and / or can include one or more transmembrane domains described herein. In one embodiment, the transmembrane domain includes a CD8 transmembrane domain. In one embodiment, the transmembrane domain includes a CD8 hinge region and a CD8 transmembrane domain. In some embodiments, the subject CAR includes a CD8 hinge region having the amino acid sequence described below:

[0379] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 86), which may be encoded by the nucleic acid sequence described below:

[0380] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 87).

[0381] Those skilled in the art will appreciate the permissible variations in the transmembrane domain while maintaining its intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 86. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region comprising an amino acid sequence recited in SEQ ID NO: 86.

[0382] In some embodiments, the subject 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 87. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region encoded by a nucleic acid sequence recited in SEQ ID NO: 87.

[0383] In some embodiments, the subject CAR comprises a CD8 hinge region having the amino acid sequence recited below: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 88),

[0384] It can be encoded by the nucleic acid sequence described below:

[0385] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 89).

[0386] Those skilled in the art will appreciate the permissible variations in transmembrane domains while maintaining their intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 88. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 transmembrane domain comprising an amino acid sequence recited in SEQ ID NO: 88.

[0387] In some embodiments, the subject 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 89. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 transmembrane domain encoded by a nucleic acid sequence recited in SEQ ID NO: 89.

[0388] In some embodiments, the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane domain having the amino acid sequence recited below:

[0389] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:90),

[0390] It can be encoded by the nucleic acid sequence described below:

[0391] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 91).

[0392] Those skilled in the art will appreciate the permissible variations in the transmembrane domain while maintaining its intended function. For example, in some embodiments, the subject CARs of the present invention comprise 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 90. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain comprising the amino acid sequence recited in SEQ ID NO:90.

[0393] In some embodiments, the subject CAR of the present invention comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain, the CD8 hinge region and the CD8 transmembrane domain being 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 recited in SEQ ID NO: 91. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain, the CD8 hinge region and the CD8 transmembrane domain being encoded by a nucleic acid sequence recited in SEQ ID NO: 91.

[0394] Between the extracellular domain and the transmembrane domain of CAR or between the intracellular domain and the transmembrane domain of CAR, a spacer domain can be incorporated. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that links the transmembrane domain to the extracellular domain or intracellular domain function in a polypeptide chain. The spacer domain can include up to 300 amino acids, such as 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer domain can be a short oligopeptide or polypeptide linker, such as, a length between 2 and 10 amino acids. For example, a glycine-serine doublet provides a special suitable linker between the transmembrane domain and the intracellular signaling domain of the subject CAR.

[0395] Intracellular signaling domain

[0396] The subject CAR of the present invention also includes 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 of the effector functions of cells (e.g., immune cells) expressing CAR. The intracellular signaling domain transduces effector function signals and guides cells (e.g., immune cells) to perform their specialized functions, such as damaging and / or destroying target cells.

[0397] Examples of intracellular domains used in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a co-stimulatory molecule, and any molecules that act synergistically to initiate signal transduction in T cells, as well as any derivatives or variants of these elements and any synthetic sequences with the same functional capability.

[0398] Examples of intracellular signaling domains include, but are not limited to, the zeta chain of the T cell receptor complex or any homolog thereof, such as the eta chain, FcsRIγ and β chains, MB 1 (Ig) 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 embodiment, the intracellular signaling domain can be human CD3 zeta chain, FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0399] In one embodiment, the intracellular signaling domain of the CAR includes any portion of one or more costimulatory molecules, such as at least one signaling domain from CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof with the same functional capability, and any combination thereof.

[0400] Other examples of intracellular domains include fragments or domains from one or more molecules or receptors including, but not limited to, TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP10, DAP 12, 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 to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD 11a, LFA-1, ITGAM, CD lib, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD 96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), 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 with the same functional ability, and combinations thereof.

[0401] Additional examples of intracellular domains include, but are not limited to, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins, including CD3, B7 family co-stimulatory, 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 and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domain (B7-H6) of NKp30 (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0402] The intracellular signaling domain suitable for use in the subject CAR of the present invention includes any desired signaling domain that provides different and detectable signals (e.g., by increasing the production of one or more cytokines by cells; changes in target gene transcription; changes in protein activity; changes in cell behavior (e.g., cell death); cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses; etc.) in response to the activation of CAR (i.e., activated by antigens and dimerizers). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif described below. In some embodiments, the intracellular signaling domain includes a DAP10 / CD28 type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to a membrane-bound CAR, but diffuses in the cytoplasm.

[0403] The intracellular signaling domain suitable for use in the subject CAR of the present invention includes an intracellular signaling polypeptide containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the ITAM motif is repeated twice in 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 the subject CAR includes 3 ITAM motifs.

[0404] 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, FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0405] Suitable intracellular signaling domains can be portions containing ITAM motifs derived from polypeptides containing ITAM motifs. For example, suitable intracellular signaling domains can be domains containing ITAM motifs from any protein containing ITAM motifs. Thus, suitable intracellular signaling domains do not need to contain the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fcε receptor Iγ chain), CD3D (CD3δ), CD3E (CD3ε), CD3G (CD3γ), CD3Z (CD3ζ), and CD79A (antigen receptor complex-associated protein α chain).

[0406] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activated protein 12; KAR-related protein; TYRO protein tyrosine kinase binding protein; killer-activated receptor-associated protein; killer-activated receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fcε receptor I gamma chain; Fc receptor gamma-chain; fc-εRI-γ; fcRγ; fceR1γ; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-Δ; T3D; CD3 antigen, delta subunit; CD3δ; 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 embodiment, 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 embodiment, 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-Γ, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T-cell surface glycoprotein CD3 ζ chain (also known as CD3Z, T-cell receptor T3 ζ chain, CD247, CD3-Z, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; ig-α; 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 includes a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises a cytoplasmic signaling domain of human CD3 ζ.

[0407] While the entire intracellular signaling domain can generally be employed, in many cases it is not necessary to use the entire chain. With respect to the use of truncated portions of the intracellular signaling domain, such truncated portions can be used in place of the entire chain, as long as they transduce the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal.

[0408] The intracellular signaling domains described herein can be combined with any antigen binding domain described herein, any transmembrane structure described herein, or any other domain described herein that can be included in a CAR.

[0409] In one embodiment, the intracellular domain of a subject CAR comprises a 4-1BB domain comprising the amino acid sequence recited below:

[0410] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 92), which may be encoded by the nucleic acid sequence described below:

[0411] AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 93),

[0412] or encoded by the nucleic acid sequence described below:

[0413] AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 94).

[0414] Those skilled in the art will appreciate the permissible variations of the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 92. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain comprising an amino acid sequence recited in SEQ ID NO: 92.

[0415] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain, the 4-1BB domain being 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 recited in SEQ ID NO: 93 or 94. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain, the 4-1BB domain being encoded by a nucleic acid sequence recited in SEQ ID NO: 93 or 94.

[0416] In one embodiment, the intracellular domain of a subject CAR comprises an ICOS domain comprising the amino acid sequence recited below:

[0417] TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL(SEQ ID NO:203),

[0418] It can be encoded by the nucleic acid sequence described below:

[0419] ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGGTGACCCTA (SEQ ID NO: 204).

[0420] Those skilled in the art will appreciate the permissible variations in the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 203. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain comprising the amino acid sequence recited in SEQ ID NO: 203.

[0421] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising an ICOS domain, the ICOS domain being 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: 204. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain, the ICOS domain being encoded by a nucleic acid sequence set forth in SEQ ID NO: 204.

[0422] In one embodiment, the intracellular domain of a subject CAR comprises a variant ICOS domain comprising the amino acid sequence recited below:

[0423] TKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTL(SEQ ID NO:95),

[0424] It can be encoded by the nucleic acid sequence described below:

[0425] ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGGTGACCCTA (SEQ ID NO:96).

[0426] The variant ICOS domain is also referred to herein as ICOS(YMNM).

[0427] Those skilled in the art will appreciate the permissible variations in the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 95. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain comprising the amino acid sequence recited in SEQ ID NO: 95.

[0428] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising an ICOS domain, the ICOS domain being 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 recited in SEQ ID NO: 96. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain, the ICOS domain being encoded by a nucleic acid sequence recited in SEQ ID NO: 96.

[0429] In one embodiment, the intracellular domain of a subject CAR comprises a CD3 zeta domain comprising the amino acid sequence recited below:

[0430] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:97),

[0431] It can be encoded by the nucleic acid sequences described below:

[0432] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:98),

[0433] or by the nucleic acid sequences described below:

[0434] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:99)。

[0435] Those skilled in the art will appreciate the permissible variations of the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 97. In one embodiment, the CAR comprises an intracellular domain comprising a CD3 zeta domain comprising the amino acid sequence recited in SEQ ID NO: 97.

[0436] In some embodiments, the 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 98 or 99. In one embodiment, the CAR comprises an intracellular domain comprising a CD3 zeta domain encoded by a nucleic acid sequence recited in SEQ ID NO: 98 or 99.

[0437] The CD3ζ domain may include the amino acid sequence described below:

[0438] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 100),

[0439] It can be encoded by the nucleic acid sequence described below:

[0440] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCTCGC(SEQ ID NO:101).

[0441] Those skilled in the art will appreciate the permissible variations in the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 100. In one embodiment, the subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain comprising an amino acid sequence recited in SEQ ID NO: 100.

[0442] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain being 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 recited in SEQ ID NO: 101. In one embodiment, the subject CAR of the present invention comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain being encoded by a nucleic acid sequence recited in SEQ ID NO: 101.

[0443] In one embodiment, the CAR comprises an intracellular domain of a CD3ζ domain comprising the amino acid sequence set forth in SEQ ID NO:97 or 100.

[0444] In an exemplary embodiment, the intracellular domain of a subject CAR comprises a 4-1BB domain and a CD3 zeta domain comprising the amino acid sequence recited below:

[0445] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 102), which may be encoded by the nucleic acid sequence described below:

[0446] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:103),

[0447] or encoded by the nucleic acid sequence described below:

[0448] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO:104).

[0449] Those skilled in the art will appreciate the permissible variations of the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain and a CD3 ζ 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 102. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain and a CD3ζ domain, wherein the 4-1BB domain and the CD3ζ domain comprise the amino acid sequence recited in SEQ ID NO:102.

[0450] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, wherein the 4-1BB domain and the CD3 zeta domain are 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 recited in SEQ ID NO: 103 or 104. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain and a CD3ζ domain, wherein the 4-1BB domain and the CD3ζ domain are encoded by the nucleic acid sequence recited in SEQ ID NO: 103 or 104.

[0451] In an exemplary embodiment, the intracellular domain of a subject CAR comprises an ICOS domain and a CD3 zeta domain comprising the amino acid sequence recited below:

[0452] TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 205),

[0453] It can be encoded by the nucleic acid sequence described below:

[0454] ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC CGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGCCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQID NO:206).

[0455] Those skilled in the art will appreciate the permissible variations in the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, wherein the ICOS domain and the CD3 zeta domain 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 recited in SEQ ID NO: 205. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain and a CD3ζ domain, wherein the ICOS domain and the CD3ζ domain comprise the amino acid sequence set forth in SEQ ID NO:205.

[0456] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, wherein the ICOS domain and the CD3 zeta domain are 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 recited in SEQ ID NO: 206. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, wherein the ICOS domain and the CD3 zeta domain are encoded by a nucleic acid sequence recited in SEQ ID NO: 206.

[0457] In an exemplary embodiment, the intracellular domain of a subject CAR comprises a variant ICOS domain and a CD3 zeta domain comprising the amino acid sequence recited below:

[0458] TKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 207),

[0459] It can be encoded by the nucleic acid sequence described below:

[0460] ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC CGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGCCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQID NO:208).

[0461] Those skilled in the art will appreciate the permissible variations in the intracellular domain while maintaining its intended function. For example, in some embodiments, the subject CARs of the present invention comprise 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%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID NO: 207. In one embodiment, the 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.

[0462] In some embodiments, the subject CAR of the present invention comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain being 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 recited in SEQ ID NO: 208. In one embodiment, the CAR comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain being encoded by a nucleic acid sequence recited in SEQ ID NO: 208.

[0463] CAR sequence

[0464] The subject CAR of the present invention can be selected from 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.

[0465] In one embodiment, the subject CAR of the present invention is J591 murine PSMA-CAR. In one embodiment, the J591 murine PSMA-CAR comprises the amino acid sequence described below:

[0466] MALPVTALLLPLALLLHAARPGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGGGSEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSASSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:105),

[0467] It can be encoded by the nucleic acid sequence described below:

[0468]

[0469] In one embodiment, the subject CAR of the present invention is a humanized PSMA-CAR, such as a humanized J591 PSMA-CAR. In such embodiments, the humanized PSMA-CAR comprises any of the heavy and light chain variable regions disclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1. For example, the humanized PSMA-CAR of the present invention may comprise an scFv comprising any of the heavy and light chain variable regions disclosed therein, see, e.g., the sequences recited in Table 19 of the present disclosure.

[0470] In one embodiment, the subject CAR of the present invention is 1C3 human PSMA-CAR. In one embodiment, 1C3 human PSMA-CAR comprises the amino acid sequence described below:

[0471] MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVPW GSRYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQFNSYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:107),

[0472] It can be encoded by the nucleic acid sequence described below:

[0473]

[0474] In one embodiment, the subject CAR of the present invention is 2A10 human PSMA-CAR. In one embodiment, the 2A10 human PSMA-CAR comprises the amino acid sequence described below:

[0475] MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQTG FLWSSDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQ QFNSYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:109),

[0476] It can be encoded by the nucleic acid sequence described below:

[0477]

[0478] In one embodiment, the subject CAR of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR comprises a 4-1BB domain and a CD3ζ domain, wherein the 4-1BB domain and the CD3ζ domain comprise the amino acid sequence described below:

[0479] MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTG FLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ FNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:111),

[0480] It can be encoded by the nucleic acid sequence described below:

[0481]

[0482] In one embodiment, the subject CAR of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR comprises an ICOS domain and a CD3 ζ domain, wherein the ICOS domain and the CD3 ζ domain comprise the amino acid sequence described below:

[0483] MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQT GFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRL TDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:209),

[0484] It can be encoded by the nucleic acid sequence described below:

[0485]

[0486] In one embodiment, the subject CAR of the present invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR comprises a variant ICOS domain and a CD3 ζ domain, wherein the variant ICOS domain and the CD3 ζ domain comprise the amino acid sequence described below:

[0487] MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQT GFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMNMRAVNTAKKSRL TDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:211),

[0488] It can be encoded by the nucleic acid sequence described below:

[0489]

[0490] In one embodiment, the subject CAR of the present invention is 2C6 human PSMA-CAR. In one embodiment, 2C6 human PSMA-CAR comprises the amino acid sequence described below:

[0491] MALPVTALLLPLALLLHAARPEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASPGYT SSWTSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ QRSNWPLFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:113),

[0492] It can be encoded by the nucleic acid sequence described below:

[0493]

[0494] Those skilled in the art are aware of the permissible variations in the sequence of a subject CAR while maintaining its function.

[0495] For example, in some embodiments, the subject 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%, 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.

[0496] For example, in some embodiments, the subject CAR of the present invention is a humanized J591 PSMA-CAR. The humanized J591 PSMA-CAR comprises a humanized J591 PSMA binding domain comprising a heavy chain and a light chain variable region selected from any of the heavy chain and light chain variable region sequences recited in Table 19. In some embodiments, the humanized J591 PSMA-CAR comprises a 4-1BB domain and a CD3 zeta domain.

[0497] For example, in some embodiments, the subject 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%, 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.

[0498] For example, in some embodiments, the subject CAR 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%, 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.

[0499] For example, in some embodiments, the subject 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, wherein the 4-1BB domain and the CD3 zeta domain 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 recited 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, the 4-1BB domain and the CD3 zeta domain comprising 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, the ICOS domain and the 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%, 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 a variant ICOS domain and a CD3 zeta domain comprising 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 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%, 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 comprising the amino acid sequence set forth in SEQ ID NO: 211. For example, in some embodiments, the subject 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%, 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.

[0500] In some embodiments, the subject CAR of the present invention is a J591 murine 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%, 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 murine PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 106.

[0501] For example, in some embodiments, the subject CAR 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%, 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, the subject 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%, 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, the subject 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, the 4-1BB domain and the CD3 zeta domain being 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, the 4-1BB domain and the CD3 zeta domain being encoded by a 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, the ICOS domain and the CD3 zeta domain being 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, the ICOS domain and the CD3 zeta domain being encoded by a 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, the variant ICOS domain and CD3 zeta domain being 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 being encoded by a nucleic acid sequence set forth in SEQ ID NO: 212. For example, in some embodiments, the subject CAR of the present invention 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%, 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.

[0502] In certain embodiments, the subject CAR of the present invention may include any one of the amino acid sequences corresponding to SEQ ID NOs: 209, 211, or 227-236.

[0503]

[0504]

[0505]

[0506]

[0507]

[0508] Therefore, the present invention provides modified immune cells or precursor cells thereof, such as modified T cells, comprising a chimeric antigen receptor (CAR) having affinity for prostate membrane antigen (PSMA) on target cells (e.g., prostate cancer cells). In some embodiments, CAR comprises a PSMA binding domain. In some embodiments, CAR comprises a murine PSMA binding domain. In one embodiment, CAR comprises a J591 murine PSMA binding domain. In one embodiment, CAR comprises a humanized J591 PSMA binding domain. In some embodiments, CAR comprises a human PSMA binding domain. In some embodiments, CAR comprises a human PSMA binding domain selected from the following: 1C3, 2A10, 2F5, and 2C6 human PSMA binding domains.

[0509] Thus, the subject 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.

[0510] Therefore, the subject CAR of the present invention includes a PSMA binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, CAR includes a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a 4-1BB domain. In one embodiment, CAR includes a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a CD3 ζ domain. In one embodiment, CAR includes a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a 4-1BB domain and a CD3 ζ domain.

[0511] C. Dominant negative receptors and switch receptors

[0512] The present invention provides compositions and methods of modified immune cells or their precursors, such as modified T cells, comprising dominant negative receptors and / or conversion receptors. Therefore, in some embodiments, the immune cells have been genetically modified to express dominant negative receptors and / or conversion receptors. As used herein, the term "dominant negative receptor" refers to a molecule designed to reduce the effect of negative signal transduction molecules (e.g., the effect of negative signal transduction molecules on the modified immune cells of the present invention). The dominant negative receptors of the present invention can utilize the extracellular domain associated with the negative signal to bind to negative signal transduction molecules, such as TGF-β or PD-1, and reduce the effect of negative signal transduction molecules. Such dominant negative receptors are described herein. For example, a modified immune cell comprising a dominant negative receptor can bind to a negative signal transduction molecule in the microenvironment of the modified immune cell and reduce the effect that the negative signal transduction molecule may have on the modified immune cell.

[0513] In addition to reducing the effects of negative signal transduction molecules, the switch receptors of the present invention can also be designed to convert negative signals into positive signals by including an intracellular domain associated with a positive signal. Described herein are switch receptors designed to convert negative signals into positive signals. Thus, the switch receptors include an extracellular domain associated with a negative signal and / or an intracellular domain associated with a positive signal.

[0514] Tumor cells produce an immunosuppressive microenvironment, which is used to protect tumor cells from being recognized and eliminated by the immune system. This immunosuppressive microenvironment may limit the effect 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 to further suppress the immune response. In the context of prostate cancer, T cell immunosuppression due to TGFβ has been previously described (Donkor et al., 2011; Shalapour et al., 2015). In order to reduce the immunosuppressive effect of TGFβ, immune cells can be modified to express dominant negative receptors that are dominant negative receptors for TGF-β.

[0515] 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 the wild-type TGF-β receptor. In some embodiments, the dominant negative receptor is a truncated dominant negative variant of the TGF-β type II receptor (TGFβRII-DN). In one embodiment, TGFβRII-DN comprises the amino acid sequence described below:

[0516] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSSG(SEQ ID NO:115),

[0517] It can be encoded by the nucleic acid sequence described below:

[0518] (SEQ ID NO: 116).

[0519] Those skilled in the art will appreciate the permissible variations in the sequence of TGFβRII-DN while maintaining its intended function. For example, in some embodiments, the 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%, 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.

[0520] In some embodiments, the dominant negative receptor of the present invention is 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%, 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 TGFβRII-DN encoded by the nucleic acid sequence set forth in SEQ ID NO: 116.

[0521] In one embodiment, a switch receptor suitable for use in the present invention is the PD1-CTM-CD28 receptor. When expressed in a cell, the PD1-CTM-CD28 receptor converts a negative PD1 signal into a positive CD28 signal. 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 comprises the amino acid sequence described below:

[0522] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTY LCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:117),

[0523] It can be encoded by the nucleic acid sequence described below:

[0524] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:118)。

[0525] Those skilled in the art will appreciate that variations in the PD1-CTM-CD28 receptor are permissible while maintaining its intended biological activity (e.g., converting a negative PD1 signal into a positive CD28 signal when expressed in a cell). Thus, the PD1-CTM-CD28 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 to the PD1-CTM-CD28 receptor amino acid sequence recited in SEQ ID NO: 117. Thus, the PD1-CTM-CD28 receptor of the present invention can be 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 PD1-CTM-CD28 receptor nucleic acid sequence recited in SEQ ID NO:118.

[0526] In one embodiment, a switch receptor suitable for use in the present invention is the PD1-PTM-CD28 receptor. When expressed in a cell, the PD1-PTM-CD28 receptor converts a negative PD1 signal into a positive CD28 signal. 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 comprises the amino acid sequence described below:

[0527] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDS GTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:119),

[0528] It can be encoded by the nucleic acid sequence described below:

[0529] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:120).

[0530] Those skilled in the art will appreciate the permissible variations of the PD1-PTM-CD28 receptor while maintaining its intended biological activity (e.g., converting a negative PD1 signal into a positive CD28 signal when expressed in a cell). Thus, the PD1-PTM-CD28 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%, at least 99% sequence identity to the PD1-PTM-CD28 receptor amino acid sequence recited in SEQ ID NO: 119. Thus, the PD1-PTM-CD28 receptor of the present invention can be 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 PD1-PTM-CD28 receptor nucleic acid sequence recited in SEQ ID NO: 120.

[0531] In one embodiment, a switch receptor suitable for use in the present invention is PD1 A132L -PTM-CD28 receptor. When expressed in cells, PD1 A132L -PTM-CD28 receptor converts negative PD1 signal into positive CD28 signal. It was found that a point mutation at amino acid position 132 of PD1 (alanine replaced by leucine) (A132L) increased its affinity for PD-L1 by two-fold (see, e.g., Zhang et al., Immunity (2004) 20(3), 337-347). A132L -PTM-CD28 receptor includes a variant of the PD1 extracellular domain, the PD1 transmembrane domain, and the CD28 cytoplasmic domain with an amino acid substitution at position 132 (A132L). In one embodiment, the PD1 A132L-PTM-CD28 receptor comprises the amino acid sequence described below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 121),

[0532] It can be encoded by the nucleic acid sequence described below:

[0533] (SEQ ID NO: 122).

[0534] Those skilled in the art know that PD1 A132L -PTM-CD28 receptors while maintaining their intended biological activity (e.g., converting a negative PD1 signal into a positive CD28 signal when expressed in a cell). A132L -PTM-CD28 receptor may include an amino acid sequence that is identical to the PD1 sequence described in SEQ ID NO: 121. A132L-PTM-CD28 receptor 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. Therefore, the PD1 of the present invention has a sequence identity of 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%. A132L -PTM-CD28 receptor can be encoded by a nucleic acid sequence that is identical to the PD1 sequence described in SEQ ID NO: 122. A132L -PTM-CD28 receptor 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.

[0535] In one embodiment, the conversion receptor suitable for use in the present invention is the PD1-4-1BB receptor. When expressed in a cell, the PD1-4-1BB receptor (also referred to herein as PD1-BB) converts a negative PD1 signal into a positive 4-1BB signal. In one embodiment, the PD1-4-1BB receptor comprises the amino acid sequence described below:

[0536] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:213),

[0537] It can be encoded by the nucleic acid sequence described below:

[0538] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:214)。

[0539] Those skilled in the art will appreciate the permissible variations of the PD1-4-1BB receptor while maintaining its intended biological activity (e.g., converting a negative PD1 signal into a positive 4-1BB signal when expressed in a cell). Thus, the 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%, at least 99% sequence identity to the PD1-4-1BB receptor amino acid sequence recited in SEQ ID NO: 213. Therefore, the PD1-4-1BB receptor of the present invention can be 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 with the PD1-4-1BB receptor nucleic acid sequence recited in SEQ ID NO: 214.

[0540] In one embodiment, a switch receptor suitable for use in the present invention is PD1 A132L -4-1BB receptor. When expressed in cells, PD1 A132L -4-1BB receptor (also referred to herein as PD1*BB) converts the negative PD1 signal into a positive 4-1BB signal. A132L -4-1BB receptor comprises the amino acid sequence described below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 215),

[0541] It can be encoded by the nucleic acid sequence described below:

[0542] (SEQ ID NO: 216).

[0543] Those skilled in the art know that PD1 A132L -4-1BB receptors while maintaining their intended biological activity (e.g., converting negative PD1 signals into positive 4-1BB signals when expressed in cells). A132L -4-1BB receptor may include such an amino acid sequence, which has the same amino acid sequence as the PD1 described in SEQ ID NO: 215. A132L-4-1BB receptor amino 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%, at least 99% sequence identity. Therefore, the PD1 of the present invention has a sequence identity of 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%. A132L The -4-1BB receptor may be encoded by a nucleic acid sequence that is identical to the PD1 sequence described in SEQ ID NO: 216. A132L -4-1BB receptor 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.

[0544] In one embodiment, a switch receptor suitable for use in the present invention is a TGFβR-IL12Rβ1 receptor. When expressed in a cell, the TGFβR-IL12Rβ1 receptor converts a negative TGF-β signal into a positive IL-12 signal. In one embodiment, the TGFβR-IL12Rβ1 receptor comprises the amino acid sequence described below:

[0545] MEAAVAAPRPRLLLLVLAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGL GPVELAAVIAGPVCFVCISLMLMVYIRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM(SEQ ID NO:123),

[0546] It can be encoded by the nucleic acid sequence described below:

[0547] ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCGGCGGCGGCGGCGGCGGCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCAGGGCCGCACGGCACCTGTGCCCGCCGCTGCCCACACCCTGTGCCAGCTCCGCCATTGAGTTCCCTGGAGGGAAGGAGACTTGGCAGTGGATCAACCCAGTGGACTTCCAGGAAGAGGCATCCCTGCAGGAGGCCCTGGTGGTAGAGATGTCCTGGGACAAAGGCGAGAGGACTGAGCCTCTCGAGAAGACAGAGCTACCTGAGGGTGCCCCTGAGCTGGCCCTGGATACAGAGTTGTCCTTGGAGGATGGAGACAGGTGCAAGGCCAAGATG(SEQ ID NO:124)。

[0548] Those skilled in the art will appreciate that variations in the TGFβR-IL12Rβ1 receptor are permissible while maintaining its intended biological activity (e.g., converting a negative TGF-β signal into a positive IL-12 signal when expressed in a cell). Thus, the TGFβR-IL12Rβ1 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 to the TGFβR-IL12Rβ1 receptor amino acid sequence set forth in SEQ ID NO: 123. Thus, the TGFβR-IL12Rβ1 receptor of the present invention can be 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 TGFβR-IL12Rβ1 receptor nucleic acid sequence recited in SEQ ID NO:124.

[0549] In one embodiment, a switch receptor suitable for use in the present invention is a TGFβR-IL12Rβ2 receptor. When expressed in a cell, the TGFβR-IL12Rβ2 receptor converts a negative TGF-β signal into a positive IL-12 signal. In one embodiment, the TGFβR-IL12Rβ2 receptor comprises the amino acid sequence described below:

[0550] MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML(SEQ ID NO:125), which can be encoded by the nucleic acid sequence described below:

[0551]

[0552] Those skilled in the art will appreciate that variations in the TGFβR-IL12Rβ2 receptor are permissible while maintaining its intended biological activity (e.g., converting a negative TGF-β signal into a positive IL-12 signal when expressed in a cell). Thus, the TGFβR-IL12Rβ2 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 to the TGFβR-IL12Rβ2 receptor amino acid sequence set forth in SEQ ID NO: 125. Thus, the TGFβR-IL12Rβ2 receptor of the present invention can be 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 TGFβR-IL12Rβ2 receptor nucleic acid sequence recited in SEQ ID NO:126.

[0553] In one embodiment, a switch receptor suitable for use in the present invention is a TIM3-CD28 receptor. When expressed in a cell, the TIM3-CD28 receptor converts a negative TIM-3 signal into a positive CD28 signal. In one embodiment, the TIM3-CD28 receptor comprises the following amino acid sequence: MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 127),

[0554] It can be encoded by the nucleic acid sequences described below:

[0555] ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:128).

[0556] Those skilled in the art will appreciate the permissible variations of the TIM3-CD28 receptor while maintaining its intended biological activity (e.g., converting a negative TIM-3 signal into a positive CD28 signal when expressed in a cell). Thus, the TIM3-CD28 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 to the TIM3-CD28 receptor amino acid sequence recited in SEQ ID NO: 127. Thus, the TIM3-CD28 receptor of the present invention can be 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 TIM3-CD28 receptor nucleic acid sequence recited in SEQ ID NO: 128.

[0557] Other suitable dominant negative receptors and switch receptors for use in the present invention are described in PCT Publication No. WO2013019615A2, the disclosure of which is incorporated herein by reference.

[0558] D. Bispecific Antibodies

[0559] The present invention provides compositions and methods of modified immune cells or their precursors, such as modified T cells, comprising nucleic acids encoding bispecific antibodies. Thus, in some embodiments, the immune cells have been genetically modified to express bispecific antibodies. As used herein, "bispecific antibodies" refer to antibodies that have binding specificity for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced using the co-expression of two immunoglobulin heavy chain / light chain pairs. See, e.g., Milstein et al. (1983) Nature 305:537-39. Alternatively, bispecific antibodies can be prepared using chemical bonding. 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.

[0560] In certain embodiments, the modified cells of the invention include a CAR with affinity for prostate membrane antigen (PSMA) on a target cell and a bispecific antibody. In certain embodiments, the modified cells of the invention secrete bispecific antibodies.

[0561] 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, a bispecific antibody comprises an antigen-binding domain that comprises a first and a second single-chain variable fragment (scFv) molecule. 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.

[0562] In one embodiment, bispecific antibodies include specificity for at least one antigen on activated T cells.Activated T cell antigens include antigens found on the surface of the T cell that can activate another cell.Activated T cell antigens can be combined with costimulatory molecules.Costimulatory molecules are cell surface molecules in addition to antigen receptors or their ligands, which are required for the effective response of lymphocytes to antigens. The example of activated T cell antigens can include but is 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 specifically bound to CD83, or a fragment thereof. In some embodiments, bispecific antibodies include specificity for T cell antigen CD28.

[0563] Other costimulatory elements are also within the scope of the present invention. In these examples, bispecific antibodies recognize T cell antigens and can be referred to as bispecific T cell adaptors (BiTEs). However, the present invention is not limited to the use of any specific bispecific antibodies. Instead, any bispecific antibody or BiTE can be used. Bispecific antibodies or BiTE molecules can also be expressed as soluble proteins that are specific for an antigen associated with at least one target cell.

[0564] 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 making human and humanized antibodies are described elsewhere herein.

[0565] In some embodiments, the bispecific antibody comprises more than one antigen binding domain, wherein at least one antigen binding domain binds to a negative signal transduction molecule (e.g., a negative signal transduction molecule that can be found in the microenvironment of a cell that secretes the bispecific antibody) or its interacting partner (e.g., a receptor). In some embodiments, at least one antigen binding domain of a bispecific antibody binds to TGF-β or its interacting partner (e.g., a receptor). In some embodiments, at least one antigen binding domain of a bispecific antibody binds to PD-1 or its interacting partner. In one embodiment, at least one antigen binding domain of a bispecific antibody binds to TGF-βR. In another embodiment, at least one antigen binding domain of a bispecific antibody binds to PD-L1.

[0566] In some embodiments, the 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, the bispecific antibody of the invention 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 by reference in their entirety.

[0567] In some embodiments, the bispecific antibody includes at least one antigen binding domain that binds to PD-L1. For example, the bispecific antibodies of the present disclosure may include, but are not limited to, the PD-L1 binding domains derived from 10A5, 13G4, or 1B12 described in PCT Publication WO2007005874A2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the bispecific antibody includes at least one antigen binding domain that binds to a TGF-β receptor, such as TGFβRII. For example, the bispecific antibodies of the present disclosure may include, but are not limited to, the TGFβRII binding domains derived from TGF1 or TGF3 disclosed in U.S. Patent No. 8,147,834, the contents of which are incorporated herein by reference in their entirety.

[0568] Thus, in one embodiment, the bispecific antibodies of the present disclosure comprise at least one antigen binding domain that binds PD-L1 or TGFβRII and an antigen binding domain that binds CD28.

[0569] In some embodiments, the target cell antigen can be the same antigen that the T cell receptor binds or can be a different antigen. The target cell antigen includes any tumor-associated antigen (TAA) or a viral, bacterial and parasitic antigen, or any fragment thereof. The target cell antigen can include any type of ligand that limits the target cell. For example, the target cell antigen can be selected to identify a ligand that acts as a cell marker on a target cell associated with a specific disease state. Therefore, a cell marker can serve 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.

[0570] In some embodiments, the target cell antigen is an antigen identical to an activated 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 specifically bound to CD83, and a fragment thereof. In one aspect, the present invention includes nucleic acids encoding bispecific antibodies comprising bispecifics for an antigen on a target cell and an antigen on an activated T cell, wherein the T cell temporarily 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), WO 93 / 08829, and Traunecker et al, EMBO J. 10:3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made using the following techniques: by engineering electrostatic steering to make antibody Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al, Science 229:81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al, J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology to make 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. 148(5):1547-1553 (1992)). al, J. Immunol, 152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991). Also included herein are engineered antibodies having three or more functional antigen-binding sites (including "Octopus antibodies") (see, e.g., US 2006 / 0025576A1). Bispecific antibodies can be constructed by linking two different antibodies or portions thereof. For example, a bispecific antibody can include Fab, F(ab')2, Fab', scFv, and sdAb from two different antibodies.

[0571] The bispecific antibodies of the present invention include bispecific antibodies with affinity for PD-L1 and CD28. In one embodiment, the 13G4-1211 PD-L1 / CD28 bispecific antibody of the present invention comprises the following amino acid sequence: MGWSCIILFLVATATGVHSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWF LDYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTT VTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEI(SEQ ID NO:129),

[0572] It can be encoded by the nucleic acid sequence described below:

[0573]

[0574] Those skilled in the art will appreciate the permissible variations of the 13G4-1211 PD-L1 / CD28 bispecific antibody while maintaining its intended biological activity (e.g., binding to PD-L1 and CD28). Thus, the 13G4-1211 PD-L1 / CD28 bispecific antibody 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 to the 13G4-1211 PD-L1 / CD28 bispecific antibody amino acid sequence recited in SEQ ID NO: 129. Therefore, the 13G4-1211 PD-L1 / CD28 bispecific antibody of the present invention can be 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 13G4-1211 PD-L1 / CD28 bispecific antibody nucleic acid sequence recited in SEQ ID NO: 130.

[0575] The bispecific antibodies of the present invention include bispecific antibodies with affinity for PD-L1 and CD28. In one embodiment, the 10A5-1412 PD-L1 / CD28 bispecific antibody of the present invention comprises the following amino acid sequence: MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWF DYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTT VTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEI(SEQ ID NO:131),

[0576] It can be encoded by the nucleic acid sequence described below:

[0577]

[0578] Those skilled in the art will appreciate the permissible variations of the 10A5-1412 PD-L1 / CD28 bispecific antibody while maintaining its intended biological activity (e.g., binding to PD-L1 and CD28). Thus, the 10A5-1412 PD-L1 / CD28 bispecific antibody 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 to the 10A5-1412 PD-L1 / CD28 bispecific antibody amino acid sequence recited in SEQ ID NO: 131. Thus, the 10A5-1412 PD-L1 / CD28 bispecific antibody of the present invention can be 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 10A5-1412 PD-L1 / CD28 bispecific antibody nucleic acid sequence set forth in SEQ ID NO: 132.

[0579] The bispecific antibodies of the present invention include bispecific antibodies with affinity for PD-L1 and CD28. In one embodiment, the 1B12-1412 PD-L1 / CD28 bispecific antibody of the present invention comprises the following amino acid sequence: MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKSGGGGSQVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGM DVWGQGTVTVSSGGSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGT TVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEI(SEQ ID NO:133),

[0580] It can be encoded by the nucleic acid sequence described below:

[0581]

[0582] Those skilled in the art will appreciate the permissible variations of the 1B12-1412 PD-L1 / CD28 bispecific antibody while maintaining its intended biological activity (e.g., binding to PD-L1 and CD28). Thus, the 1B12-1412 PD-L1 / CD28 bispecific antibody 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 to the 1B12-1412 PD-L1 / CD28 bispecific antibody amino acid sequence recited in SEQ ID NO: 133. Thus, the 1B12-1412 PD-L1 / CD28 bispecific antibody of the present invention can be 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 1B12-1412 PD-L1 / CD28 bispecific antibody nucleic acid sequence recited in SEQ ID NO: 134.

[0583] The bispecific antibodies of the present invention include bispecific antibodies having affinity for TGF-β receptor type II (TGFβRII) and CD28. In one embodiment, the TGFβR-1-1412TGFβRII / CD28 bispecific antibody of the present invention comprises the amino acid sequence described below:

[0584] MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO:135),

[0585] It can be encoded by the nucleic acid sequence described below:

[0586]

[0587] Those skilled in the art will appreciate the permissible variations of the TGFβR-1-1412TGFβRII / CD28 bispecific antibody while maintaining its intended biological activity (eg, binding to TGFβRII and CD28). Thus, the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody 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 to the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody amino acid sequence recited in SEQ ID NO:135. Thus, the TGFβR-1-1412 TGFβRII / CD28 bispecific antibody of the present invention can be 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 TGFβR-1-1412 TGFβRII / CD28 bispecific antibody nucleic acid sequence recited in SEQ ID NO: 136.

[0588] The bispecific antibodies of the present invention include bispecific antibodies having affinity for TGF-β receptor type II (TGFβRII) and CD28. In one embodiment, the TGFβR-3-1412TGFβRII / CD28 bispecific antibody of the present invention comprises the amino acid sequence described below:

[0589] MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVRSFLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYSWGWIRQPPGKGLEWIGSFYYSGITYYSPSLKSRIIISEDTSKNQFSLKLSSVTAADTAVYYCASGFTMIRGALDYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO:137),

[0590] It can be encoded by the nucleic acid sequence described below:

[0591]

[0592] Those skilled in the art will appreciate the permissible variations of the TGFβR-3-1412TGFβRII / CD28 bispecific antibody while maintaining its intended biological activity (eg, binding to TGFβRII and CD28). Thus, the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody 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 TGFβR-3-1412 TGFβRII / CD28 bispecific antibody amino acid sequence recited in SEQ ID NO:137. Thus, the TGFβR-3-1412 TGFβRII / CD28 bispecific antibody of the present invention can be 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 TGFβR-3-1412 TGFβRII / CD28 bispecific antibody nucleic acid sequence recited in SEQ ID NO: 138.

[0593] 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.

[0594] E. Nucleic Acids and Expression Vectors

[0595] The present invention provides nucleic acids encoding CAR and / or dominant negative receptors and / or conversion receptors. In one embodiment, the nucleic acid of the present disclosure includes a nucleic acid sequence encoding a subject CAR of the present invention (e.g., PSMA-CAR). In one embodiment, the nucleic acid of the present disclosure includes a nucleic acid sequence encoding a dominant negative receptor and / or conversion receptor (e.g., PD1-PTM-CD28 receptor).

[0596] In some embodiments, the nucleic acids of the present disclosure provide for the production of CAR and / or dominant negative receptors and / or switch receptors as described herein in mammalian cells. In some embodiments, the nucleic acids of the present disclosure provide for the amplification of nucleic acids encoding CAR and / or dominant negative receptors and / or switch receptors.

[0597] As described herein, the subject CAR includes an antigen binding domain, a transmembrane domain, and an intracellular domain. Therefore, the present disclosure provides nucleic acids encoding the antigen binding domain, transmembrane domain, and intracellular domain of the subject CAR. As described herein, various dominant negative receptors and conversion receptors are provided. Therefore, the present invention provides nucleic acids encoding dominant negative receptors and / or conversion receptors.

[0598] In some embodiments, the nucleic acid encoding the CAR is separated from the nucleic acid encoding the dominant negative receptor and / or the switch receptor. In an exemplary embodiment, the nucleic acid encoding the CAR and the nucleic acid encoding the dominant negative receptor and / or the switch receptor are present in the same nucleic acid.

[0599] In some embodiments, the nucleic acid of the present invention includes a nucleic acid containing a CAR coding sequence and a dominant negative receptor and / or a conversion receptor coding sequence. In some embodiments, the nucleic acid of the present invention includes a nucleic acid containing a CAR coding sequence and a dominant negative receptor and / or a conversion receptor coding sequence separated by a joint. The joint used in the present invention (e.g., in the context of connecting a CAR coding sequence and a dominant negative receptor and / or a conversion receptor coding sequence) allows multiple proteins (e.g., polycistronic or bicistronic sequences) to be encoded by the same nucleic acid sequence, which is translated into a polyprotein that is dissociated into separate protein components. For example, the joint used in the nucleic acid of the present invention containing a CAR coding sequence and a dominant negative receptor and / or a conversion receptor coding sequence allows CAR and a dominant negative receptor and / or a conversion receptor to be translated into a polyprotein that is dissociated into separate CAR and dominant negative receptor and / or conversion receptor components.

[0600] In some embodiments, the linker includes a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, an "internal ribosome entry site" or "IRES" refers to an element that facilitates direct entry of internal ribosomes into the start codon (such as ATG) of a protein coding region, thereby allowing the gene to undergo cap-independent translation. Various internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRES that can be obtained 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 IRES that can be obtained from, for example, cardiovirus, rhinovirus, foot-and-mouth disease virus, HCV, Friedreich's murine leukemia virus (FrMLV) and Moloney's murine leukemia virus (MoMLV). Those skilled in the art will be able to select an IRES suitable for use in the present invention.

[0601] In some embodiments, the linker includes a nucleic acid sequence encoding a self-cleaving peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein, which dissociates into component proteins after translation. The use of the term "self-cleavage" does not mean a hydrolytic cleavage reaction. Various self-cleaving peptides or 2A peptides are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae family, such as foot-and-mouth disease virus (FMDV), equine rhinitis virus A (ERAV0), scutellaria virus (TaV) and porcine tetanus virus-1 (PTV-1), and cardioviruses such as Theilovirus 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 skilled in the art will be able to select a self-cleaving peptide suitable for use in the present invention.

[0602] In some embodiments, the nucleic acid of the present disclosure includes a nucleic acid sequence comprising a CAR coding sequence and a dominant negative receptor and / or a switch receptor coding sequence separated by a joint, and the joint includes a T2A peptide sequence. In some embodiments, the T2A peptide sequence includes the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 139), which can be encoded by the nucleic acid sequence GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 140). In some embodiments, the joint including the T2A peptide sequence can further include a spacer sequence described herein. For example, the joint including the T2A peptide sequence can further include a spacer containing the amino acid sequence SGRSGGG (SEQ ID NO: 141), which can be encoded by the nucleic acid sequence TCCGGAAGATCTGGCGGCGGA (SEQ ID NO: 142).

[0603] In some embodiments, the nucleic acids of the present disclosure include a nucleic acid sequence comprising a CAR coding sequence and a dominant negative receptor and / or switch receptor coding sequence separated by a linker, the linker comprising a F2A peptide sequence. In some embodiments, the F2A peptide sequence comprises the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 143), which can be encoded by the nucleic acid sequence GTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCG (SEQ ID NO: 144).

[0604] In some embodiments, the joint further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitous protease that is present in trans-Hokkigenia and processes its precursor before protein secretion. Furin cuts at the COOH-terminal end of its consensus recognition sequence. The consensus recognition sequence (or " furin cleavage site ") of various furins known to those skilled in the art includes but is not limited to Arg-X-Lys-Arg (SEQ ID NO:145) or Arg-X-Arg-Arg (SEQ ID NO:146), and Arg-XX-Arg (SEQ ID NO:147), such as Arg-Gln-Lys-Arg (SEQ ID NO:148), 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), wherein X1 is Lys or Arg, X2 is any naturally occurring amino acid, and X3 is Lys or Arg. One skilled in the art will be able to select an appropriate furin cleavage site for use in the present invention.

[0605] 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, and a linker comprising a nucleic acid sequence encoding furin and T2A. One 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 the furin and 2A peptide. Various 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 can include, but are not limited to, amino acid sequences including but not limited to: GGSG (SEQ ID NO: 4), GGSGG (SEQ ID NO: 5), GSGSG (SEQ ID NO: 6), GSGGG (SEQ ID NO: 7), GGGSG (SEQ ID NO: 8), GSSSG (SEQ ID NO: 9), etc. One skilled in the art will be able to select suitable spacer sequences for use in the present invention.

[0606] In some embodiments, nucleic acid of the present disclosure includes a nucleic acid sequence containing a CAR coding sequence and a dominant negative receptor and / or a switch receptor coding sequence separated by a furin-(G4S)2-T2A (F-GS2-T2A) joint. The F-GS2-T2A joint can be encoded by the nucleic acid sequence CGTGCGAAGAGGGGCGGCGGGGGCTCCGGCGGGGGAGGCAGTGAGGGCCGCGGCTCCCTGCTGACCTGCGGAGATGTAGAAGAGAACCCAGGCCCC (SEQ ID NO: 150) and can include the amino acid sequence RAKRGGGGSGGGGSEGRGSLLTCGDVEENPGP (SEQ ID NO: 151). Those skilled in the art will recognize that the joint of the present invention can include permissible sequence changes.

[0607] In some embodiments, the present invention provides nucleic acids comprising nucleic acid sequences encoding dominant negative receptors and / or conversion receptors as described herein. In some embodiments, nucleic acids include nucleic acid sequences encoding dominant negative receptors and / or conversion receptors and nucleic acid sequences encoding CAR as described herein (e.g., PSMA-CAR). In one embodiment, the nucleic acid sequences encoding dominant negative receptors and / or conversion receptors and the nucleic acid sequences encoding CAR are present on separate nucleic acids. In one embodiment, the nucleic acid sequences encoding dominant negative receptors and / or conversion receptors and the nucleic acid sequences encoding CAR are present in the same nucleic acid. In such embodiments, the nucleic acid sequences encoding dominant negative receptors and / or conversion receptors and the nucleic acid sequences encoding CAR are separated by joints as described herein.

[0608] For example, nucleic acid of the present disclosure may include a nucleic acid sequence encoding a dominant receptor, a joint, and encoding CAR. In one embodiment, the joint includes a nucleic acid sequence encoding a 2A peptide (e.g., T2A). In an exemplary embodiment, nucleic acid of the present disclosure may include a nucleic acid sequence encoding a dominant negative receptor and / or a conversion receptor separated by a joint including a nucleic acid sequence encoding T2A and a nucleic acid sequence encoding CAR.

[0609] Therefore, in one embodiment, the nucleic acid of the present disclosure includes from 5' to 3': 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, the nucleic acid of the present disclosure includes from 5' to 3' a nucleic acid sequence encoding a CAR, a CAR encoding a linker, a nucleic acid sequence encoding a dominant negative receptor and / or a switch receptor.

[0610]

[0611] In one embodiment, the CAR is a humanized J591 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the present 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, the nucleic acid of the present 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.

[0612] In one embodiment, the CAR is a humanized J591 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the present 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.

[0613] The humanized PSMA-CAR may include any of the heavy and light chain variable regions disclosed in PCT Publication Nos. WO2017212250A1 and WO2018033749A1. For example, the humanized PSMA-CAR of the present invention may include an scFv comprising any of the heavy and light chain variable regions disclosed herein. In some embodiments, the humanized J591 PSMA-CAR includes 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 described in Table 19.

[0614] In one embodiment, the CAR is a human 1C3 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present 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 human 1C3 PSMA-CAR. In one embodiment, the nucleic acid comprising, 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 human 1C3 PSMA-CAR comprises the following nucleic acid sequence:

[0615]

[0616] In one embodiment, the CAR is a human 2A10 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the present 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 human 2A10 PSMA-CAR. In one embodiment, the nucleic acid comprising, 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 human 2A10 PSMA-CAR comprises the following nucleic acid sequence:

[0617]

[0618] In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, the nucleic acid of the present 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 human 2F5 PSMA-CAR. In one embodiment, the nucleic acid comprising, 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 human 2F5 PSMA-CAR comprises the following nucleic acid sequence:

[0619]

[0620] In one embodiment, the CAR is a human 2C6 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present 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 human 2C6 PSMA-CAR. In one embodiment, the nucleic acid comprising, 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 human 2C6 PSMA-CAR comprises the following nucleic acid sequence:

[0621]

[0622] Those skilled in the art will appreciate the permissible variations in the nucleic acid sequences encoding TGFβRII-DN and PSMA-CAR. 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%, at least 99% sequence identity to the nucleic acid sequence recited in any one of SEQ ID NOs: 152-156. In one embodiment, the nucleic acid sequence encoding TGFβRII-DN and murine J591 PSMA-CAR comprises the nucleic acid sequence recited in SEQ ID NO: 152. In one embodiment, the nucleic acid sequence encoding 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 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 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 TGFβRII-DN and human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 156.

[0623] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 1C3 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', 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, from 5' to 3', 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 comprises the nucleic acid sequence described below:

[0624]

[0625] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', 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, the 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 comprises the following nucleic acid sequences:

[0626]

[0627] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', 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, the nucleic acid comprising, from 5' to 3', 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 comprises the nucleic acid sequence described below:

[0628]

[0629] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', 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, the nucleic acid comprising, from 5' to 3', 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 comprises the following nucleic acid sequence:

[0630]

[0631] Those skilled in the art will appreciate the permissible variations in nucleic acid sequences encoding PD1-CTM-CD28 and PSMA-CAR. 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%, at least 99% sequence identity to 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 recited 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.

[0632] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is human 1C3 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises from 5' to 3': encoding PD1 A132L -PTM-CD28 nucleic acid sequence, a nucleic acid sequence encoding a linker including F2A, and a nucleic acid sequence encoding human 1C3 PSMA-CAR. In one embodiment, from 5' to 3' includes: encoding PD1 A132L The nucleic acid sequence encoding the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 1C3 PSMA-CAR include the nucleic acid sequences described below:

[0633]

[0634] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is human 2A10 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises from 5' to 3': encoding PD1 A132L -PTM-CD28 nucleic acid sequence, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2A10 PSMA-CAR. In one embodiment, from 5' to 3' comprises: encoding PD1 A132L The nucleic acid sequence encoding the PTM-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding the human 2A10 PSMA-CAR include the nucleic acid sequences described below:

[0635]

[0636] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is human 2F5 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises from 5' to 3': encoding PD1 A132L -PTM-CD28 nucleic acid sequence, a nucleic acid sequence encoding a linker including F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR. In one embodiment, from 5' to 3' includes: encoding PD1 A132L The nucleic acid sequence encoding the PTM-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding the human 2F5 PSMA-CAR include the nucleic acid sequences described below:

[0637]

[0638] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is human 2C6 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises from 5' to 3': encoding PD1 A132L -PTM-CD28 nucleic acid sequence, a nucleic acid sequence encoding a linker including F2A, and a nucleic acid sequence encoding human 2C6 PSMA-CAR. In one embodiment, from 5' to 3' includes: encoding PD1 A132L The nucleic acid sequence encoding the PTM-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding the human 2C6 PSMA-CAR include the nucleic acid sequences described below:

[0639]

[0640] Those skilled in the art know that the encoding PD1 A132L -Permissible variations in the nucleic acid sequences of PTM-CD28 and PSMA-CAR. 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%, at least 99% sequence identity to the nucleic acid sequence recited in any one of SEQ ID NOs: 161-164. In one embodiment, the nucleic acid sequence encoding PD1 A132L -PTM-CD28 and human 1C3 PSMA-CAR nucleic acid sequence includes the nucleic acid sequence described in SEQ ID NO: 161. In one embodiment, encoding PD1 A132L -PTM-CD28 and human 2A10 PSMA-CAR nucleic acid sequence includes the nucleic acid sequence described in SEQ ID NO: 162. In one embodiment, encoding PD1 A132L -PTM-CD28 and human 2F5 PSMA-CAR nucleic acid sequence includes the nucleic acid sequence described in SEQ ID NO: 163. In one embodiment, encoding PD1 A132L The nucleic acid sequences of -PTM-CD28 and human 2C6 PSMA-CAR include the nucleic acid sequence set forth in SEQ ID NO: 164.

[0641] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 TIM3-CD28. In one embodiment, the CAR is a human 1C3 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', a nucleic acid sequence encoding TIM3-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, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR comprises the nucleic acid sequence described below:

[0642]

[0643] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 TIM3-CD28. In one embodiment, the CAR is a human 2A10 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR. In one embodiment, the nucleic acid comprising, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR comprises the nucleic acid sequence described below:

[0644]

[0645] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 TIM3-CD28. In one embodiment, the CAR is a human 2F5 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR. In one embodiment, the nucleic acid comprising, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR comprises the nucleic acid sequence described below:

[0646]

[0647] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 TIM3-CD28. In one embodiment, the CAR is a human 2C6 PSMA-CAR. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR. In one embodiment, the nucleic acid comprising, from 5' to 3', a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR comprises the nucleic acid sequence described below:

[0648]

[0649] Those skilled in the art will appreciate the permissible variations in nucleic acid sequences encoding TIM3-CD28 and PSMA-CAR. 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 recited in any one of SEQ ID NOs: 165-168. In one embodiment, the nucleic acid sequence encoding TIM3-CD28 and human 1C3 PSMA-CAR comprises the nucleic acid sequence recited in SEQ ID NO: 165. In one embodiment, the nucleic acid sequence encoding TIM3-CD28 and human 2A10 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 166. In one embodiment, the nucleic acid sequence encoding TIM3-CD28 and human 2F5 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 167. In one embodiment, the nucleic acid sequence encoding TIM3-CD28 and human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 168.

[0650] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 comprising an ICOS domain and a CD3 ζ domain. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', 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 comprising an ICOS domain and a CD3 ζ domain. In one embodiment, the nucleic acid comprising, from 5' to 3', 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 comprising an ICOS domain and a CD3 ζ domain comprises the following nucleic acid sequences:

[0651]

[0652] Those skilled in the art will appreciate the permissible variations in the nucleic acid sequences encoding PD1-CTM-CD28 and a human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 217. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and a human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence recited in SEQ ID NO: 217.

[0653] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', 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 comprising a variant ICOS domain and a CD3 ζ domain. Therefore, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', 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 comprising a variant ICOS domain and a CD3 ζ domain. In one embodiment, the nucleic acid comprising, from 5' to 3', 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 comprising a variant ICOS domain and a CD3 ζ domain comprises the following nucleic acid sequences:

[0654]

[0655] Those skilled in the art will appreciate the permissible variations in the nucleic acid sequences encoding PD1-CTM-CD28 and human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 218. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence recited in SEQ ID NO: 218.

[0656] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising an ICOS domain and a CD3ζ domain. Thus, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3': encoding PD1 A132L -PTM-CD28 nucleic acid sequence, encoding a linker comprising F2A nucleic acid sequence, and encoding a human 2F5PSMA-CAR nucleic acid sequence comprising an ICOS domain and a CD3ζ domain. In one embodiment, from 5' to 3' comprises: encoding PD1 A132L The nucleic acid sequence encoding the PTM-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding the human 2F5 PSMA-CAR including the ICOS domain and the CD3ζ domain include the nucleic acid sequences described below:

[0657]

[0658] Those skilled in the art know that the encoding PD1 A132L Permissible variations in the nucleic acid sequence of -PTM-CD2 and 2F5PSMA-CAR comprising an ICOS domain and a CD3ζ domain. 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 219. In one embodiment, the nucleic acid sequence encoding PD1 A132L The nucleic acid sequences of -PTM-CD28 and human 2F5 PSMA-CAR comprising an ICOS domain and a CD3ζ domain include the nucleic acid sequence set forth in SEQ ID NO: 219.

[0659] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -PTM-CD28. In one embodiment, the CAR is a human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3ζ domain. Thus, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3': encoding PD1 A132L -PTM-CD28 nucleic acid sequence, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3ζ domain. In one embodiment, from 5' to 3' comprises: encoding PD1 A132L The nucleic acid sequence encoding the PTM-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding the human 2F5 PSMA-CAR including a variant ICOS domain and a CD3ζ domain include the nucleic acid sequences described below:

[0660]

[0661] Those skilled in the art know that the encoding PD1 A132L Permissible variations in the nucleic acid sequences of -PTM-CD28 and human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3ζ domain. 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%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID NO: 220. In one embodiment, the nucleic acid sequence encoding PD1 A132L The nucleic acid sequences of -PTM-CD28 and human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3ζ domain include the nucleic acid sequence set forth in SEQ ID NO: 220.

[0662] In some embodiments, the nucleic acid of the present disclosure comprises, from 5' to 3', a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding CAR. In one embodiment, the switch receptor is PD1 A132L -4-1BB. In one embodiment, the CAR is a human 2F5PSMA-CAR comprising an ICOS domain and a CD3ζ domain. Thus, in an exemplary embodiment, the nucleic acid of the present invention comprises, from 5' to 3', a sequence encoding PD1 A132L -4-1BB nucleic acid sequence, a nucleic acid sequence encoding a linker including F2A, and a nucleic aci...

Claims

1. A modified immune cell comprising: A chimeric antigen receptor (CAR) having affinity for prostate-specific membrane antigen (PSMA) on a target cell, wherein the amino acid sequence of the CAR consists of the amino acid sequence of any one of SEQ ID NOs: 111, 209, 211, and 227-236.

2. The modified immune cell of claim 1, wherein the modified immune cell further comprises a bispecific antibody.

3. The modified immune cell of claim 2, wherein the bispecific antibody comprises a first antigen binding domain and a second antigen binding domain.

4. The modified immune cell of claim 3, wherein the first antigen binding domain binds a negative signal selected from CTLA4, PD-1, BTLA, TIM-3, and TGFβR.

5. The modified immune cell of claim 3, wherein the second antigen binding domain binds a co-stimulatory molecule. The modified immune cell of claim 5 , wherein the co-stimulatory molecule is CD28.

7. The modified immune cell according to any one of claims 1-6, wherein the modified immune cell is a modified T cell.

8. The modified immune cell of claim 7, wherein the modified T cell is an autologous cell.

9. The modified immune cell according to any one of claims 1-6, wherein the modified immune cell is a cytotoxic T lymphocyte (CTL).

10. The modified immune cell of any one of claims 1-6, wherein the modified immune cell is a natural killer (NK) cell.

11. The modified immune cell according to any one of claims 1-6, wherein the modified immune cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

12. The modified immune cell of any one of claims 1-6, wherein the modified immune cell is an autologous cell.

13. The modified immune cell according to any one of claims 1-6, wherein the modified immune cell is derived from a human.

14. The modified immune cell according to claim 7, wherein the modified T cell is derived from a human.

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