Cells expressing c-kit mutations and uses thereof

Abstract

To provide methods and compositions for enhancing immune responses against cancer and pathogens.SOLUTION: The presently disclosed subject matter provides methods and compositions for enhancing immune responses against cancer and pathogens. The presently disclosed subject matter relates to cells comprising a c-Kit mutant, e.g., a c-Kit mutant comprising an activating mutation. The cell may further comprise an antigen recognizing receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)). The presently disclosed subject matter relates to the use of cells for treatment, e.g., treatment of cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 943,032, filed December 3, 2019, the contents of which are incorporated herein by reference in their entirety and to which priority is claimed.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on December 3, 2020, is designated 0727341174_ST25 and is 57,790 bytes in size.

[0003] 1. Introduction The subject matter of the present disclosure provides methods and compositions for enhancing immune responses to cancer and pathogens.The subject matter of the present disclosure relates to cells comprising c-Kit mutants, for example, c-Kit mutants comprising activating mutations.The cells may further comprise an antigen recognition receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)).The subject matter of the present disclosure relates to the use of the cells for treatment, for example, for the treatment of cancer. [Background technology]

[0004] 2. Background of the invention Cell-based immunotherapy is a potentially curative therapy for the treatment of cancer. T cells and other immune cells can be engineered to target tumor antigens through the introduction of genetic material encoding natural or engineered T cell receptors (TCRs) specific for a selected antigen, or synthetic receptors for the antigen, called chimeric antigen receptors (CARs). Patient-engineered CAR T cells have demonstrated remarkable efficacy against a variety of liquid and solid malignancies.

[0005] CARs in clinical and preclinical development mainly use costimulatory domains, such as CD28 or 4-1BB.The persistence of these CARs, especially functional persistence, has been shown to be associated with better results.Compared with existing CARs, there is an unmet need for improved CARs that have enhanced proliferation and persistence without costimulatory domains, and / or have improved efficiency and activity. Summary of the Invention [Means for solving the problem]

[0006] 3. Summary of the Invention The presently disclosed subject matter provides cells comprising (a) an antigen-recognizing receptor that binds to an antigen, and (b) a mutant of human c-Kit. In certain embodiments, the c-Kit mutant comprises an activating mutation.

[0007] In certain embodiments, the c-Kit is human c-Kit. In certain embodiments, the activating mutation is in the intracellular domain of human c-Kit. In certain embodiments, the intracellular domain comprises amino acids 544 to 977 of human c-Kit.

[0008] In certain embodiments, the activating mutation is within amino acids 816 to 826 of human c-Kit. In certain embodiments, the activating mutation is at amino acid position 816 or amino acid position 822.

[0009] In certain embodiments, the activating mutation is within amino acids 550 to 570 of human c-Kit. In certain embodiments, the activating mutation is at amino acid position 560 of human c-Kit.

[0010] In certain embodiments, the activating mutation is selected from D816V, D816Y, D816H, D816F, N822K, V560G, or a combination thereof. In certain embodiments, the activating mutation comprises or consists of D816V.

[0011] In certain embodiments, the human c-Kit comprises or consists of the amino acid sequence set forth in SEQ ID NO:1.

[0012] In certain embodiments, the variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, or a portion thereof. In certain embodiments, the variant consists of amino acids 543 to 976 of SEQ ID NO: 2.

[0013] In certain embodiments, the c-Kit mutant is operably linked to an inducible promoter, hi certain embodiments, the inducible promoter is selected from the group consisting of a nuclear factor of activated T cells (NFAT), a transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter.

[0014] In certain embodiments, the c-Kit mutant enhances cellular persistence of the cell. In certain embodiments, the c-Kit mutant reduces apoptosis or anergy of the cell.

[0015] In certain embodiments, the antigen-recognizing receptor is exogenous or endogenous. In certain embodiments, the antigen-recognizing receptor is recombinantly expressed. In certain embodiments, the antigen-recognizing receptor is expressed from a vector. In certain embodiments, the c-Kit mutant is expressed from a vector.

[0016] In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a lymphoid cell or a myeloid cell. In certain embodiments, the lymphoid cell is selected from a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL), a γδ T cell, a tumor-infiltrating lymphocyte (TIL), a regulatory T cell, or a natural killer T (NKT) cell.

[0017] In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is mesothelin, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell. Selected from the group consisting of adhesion molecules, MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. In certain embodiments, the antigen is mesothelin.

[0018] In certain embodiments, the antigen-recognizing receptor is selected from a T cell receptor (TCR), a chimeric antigen receptor (CAR), and a TCR-like fusion molecule. In certain embodiments, the antigen-recognizing receptor is a CAR. In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide. In certain embodiments, the CAR does not comprise a costimulatory signaling region.

[0019] Furthermore, the present disclosure provides a method for generating antigen-specific immune cells. In certain embodiments, the method includes introducing into cells (a) a first nucleic acid sequence encoding an antigen-recognizing receptor that binds to an antigen; and (b) a second nucleic acid sequence encoding a c-Kit mutant containing an activating mutation. In certain embodiments, the first nucleic acid sequence is operably linked to a first promoter. In certain embodiments, the second nucleic acid sequence is operably linked to a second promoter. In certain embodiments, one or both of the first and second nucleic acid sequences are contained in a vector. In certain embodiments, the vector is a retroviral vector.

[0020] Furthermore, the present disclosure provides a composition comprising: a) a mutant of human c-Kit comprising an activating mutation; and b) an antigen-recognizing receptor that binds to an antigen. In certain embodiments, the c-Kit mutant is operably linked to a first promoter. In certain embodiments, the antigen-recognizing receptor is operably linked to a second promoter. In certain embodiments, one or both of the first and second promoters are inducible promoters. In certain embodiments, the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter. The present disclosure further provides a cell comprising the composition disclosed herein.

[0021] The present disclosure further provides a nucleic acid composition comprising (a) a first polynucleotide encoding an antigen-recognizing receptor that binds to an antigen, and (b) a second polynucleotide encoding a variant of human c-Kit comprising an activating mutation. In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the c-Kit variant. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the antigen-recognizing receptor. In certain embodiments, one or both of the first and second promoters are inducible promoters. In certain embodiments, the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter. In certain embodiments, one or both of the first and second polynucleotides are comprised in a vector. In certain embodiments, the vector is a retroviral vector. The present disclosure further provides a cell comprising the nucleic acid composition disclosed herein.

[0022] The present disclosure further provides a vector comprising the nucleic acid composition disclosed herein.The present disclosure further provides a cell comprising the vector disclosed herein.

[0023] The present disclosure further provides a pharmaceutical composition comprising the cells disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the composition further comprises a c-Kit inhibitor. In certain embodiments, the c-Kit inhibitor is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof. In certain embodiments, the pharmaceutical composition is for treating and / or preventing neoplasms, pathogen infections, or infectious diseases.

[0024] The present disclosure further provides a method for reducing tumor burden in a subject.The present disclosure also provides a method, comprising administering to a subject the cells disclosed herein or the pharmaceutical compositions disclosed herein.In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

[0025] The present disclosure further provides methods for treating and / or preventing neoplasms. In certain embodiments, the methods comprise administering to a subject a cell disclosed herein or a pharmaceutical composition disclosed herein.

[0026] The present disclosure further provides a method for extending the survival time of a subject having a neoplasm. In certain embodiments, the method comprises administering to the subject a cell disclosed herein or a pharmaceutical composition disclosed herein.

[0027] In certain embodiments, the tumor or neoplasm is a solid tumor. In certain embodiments, the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, cholangiocarcinoma, and combinations thereof. In certain embodiments, the solid tumor is mesothelioma. In certain embodiments, the solid tumor is lung cancer.

[0028] In certain embodiments, the method further comprises administering an inhibitor of c-Kit, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof.

[0029] Furthermore, the present disclosure provides a kit comprising the cells disclosed herein, the compositions disclosed herein, the nucleic acid compositions disclosed herein, or the vectors disclosed herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm, a pathogen infection, or an infectious disease. In an embodiment of the present invention, for example, the following items are provided: (Item 1) (a) an antigen-recognizing receptor that binds to an antigen; and (b) c-Kit mutants containing activating mutations Cells containing (Item 2) 2. The cell of item 1, wherein the c-Kit is human c-Kit. (Item 3) 3. The cell of item 2, wherein the activating mutation is in the intracellular domain of human c-Kit. (Item 4) 4. The cell according to item 3, wherein the intracellular region comprises amino acids 544 to 977 of human c-Kit. (Item 5) 5. The cell of any one of items 2 to 4, wherein the activating mutation is within amino acids 816 to 826 of human c-Kit. (Item 6) 6. The cell of any one of items 2 to 5, wherein the activating mutation is at amino acid position 816 or amino acid position 822. (Item 7) 5. The cell of any one of items 2 to 4, wherein the activating mutation is within amino acids 550 to 570 of human c-Kit. (Item 8) 8. The cell of any one of items 2 to 4 and 7, wherein the activating mutation is at amino acid position 560 of human c-Kit. (Item 9) 5. The cell of items 2 to 4, wherein the activating mutation is selected from D816V, D816Y, D816H, D816F, N822K, V560G, or a combination thereof. (Item 10) 10. The cell of any one of items 2 to 4 and 9, wherein the activating mutation comprises or consists of D816V. (Item 11) 11. The cell of any one of items 2 to 10, wherein the human c-Kit comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. (Item 12) 12. The cell of any one of items 1 to 11, wherein the mutant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 or a part thereof. (Item 13) 13. The cell according to any one of items 1 to 12, wherein the mutant consists of amino acids 543 to 976 of SEQ ID NO: 2. (Item 14) 14. The cell of any one of items 1 to 13, wherein the c-Kit mutant is operably linked to an inducible promoter. (Item 15) 15. The cell of item 14, wherein the inducible promoter is selected from the group consisting of a nuclear factor of activated T cells (NFAT), a transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter. (Item 16) 16. The cell of any one of items 1 to 15, wherein the c-Kit mutant enhances cellular persistence of the cell. (Item 17) 17. The cell of any one of items 1 to 16, wherein the c-Kit mutant reduces apoptosis or anergy of the cell. (Item 18) 18. The cell of any one of items 1 to 17, wherein the antigen-recognizing receptor is exogenous or endogenous. (Item 19) 19. The cell of any one of items 1 to 18, wherein the antigen-recognizing receptor is recombinantly expressed. (Item 20) 20. The cell of any one of items 1 to 19, wherein the antigen-recognizing receptor is expressed from a vector. (Item 21) 21. The cell of any one of items 1 to 20, wherein the c-Kit mutant is expressed from a vector. (Item 22) 22. The cell of any one of items 1 to 21, which is an immunoresponsive cell. (Item 23) 23. The cell of any one of items 1 to 22, which is a cell of lymphoid lineage or a cell of myeloid lineage. (Item 24) 24. The cell according to item 23, wherein the cell of the lymphoid lineage is selected from a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell. (Item 25) 25. The cell of any one of items 1 to 24, which is a T cell. (Item 26) 26. The cell of item 24 or 25, wherein the T cell is a cytotoxic T lymphocyte (CTL), a γδ T cell, a tumor-infiltrating lymphocyte (TIL), a regulatory T cell, or a natural killer T (NKT) cell. (Item 27) 27. The cell of any one of items 1 to 26, wherein the antigen is a tumor antigen or a pathogen antigen. (Item 28) 28. The cell of any one of items 1 to 27, wherein the antigen is a tumor antigen. (Item 29) the tumor antigen is mesothelin, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, 29. The cell of item 28, selected from the group consisting of MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. (Item 30) 30. The cell of item 29, wherein the antigen is mesothelin. (Item 31) 31. The cell of any one of items 1 to 30, wherein the antigen-recognizing receptor is selected from a T cell receptor (TCR), a chimeric antigen receptor (CAR), and a TCR-like fusion molecule. (Item 32) 32. The cell of any one of items 1 to 31, wherein the antigen recognition receptor is a CAR. (Item 33) 33. The cell of item 32, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling region. (Item 34) 34. The cell of paragraph 33, wherein the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. (Item 35) 35. The cell of item 34, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide. (Item 36) 34. The cell of item 33, wherein the CAR does not comprise a costimulatory signaling region. (Item 37) A method for generating an antigen-specific immunoresponsive cell, comprising the steps of introducing into a cell: (a) a first nucleic acid sequence encoding an antigen-recognizing receptor that binds to the antigen; and (b) a second nucleic acid sequence encoding a c-Kit mutant containing an activating mutation. (Item 38) 38. The method of claim 37, wherein the first nucleic acid sequence is operably linked to a first promoter. (Item 39) 39. The method of claim 37 or 38, wherein the second nucleic acid sequence is operably linked to a second promoter. (Item 40) One or both of the first and second nucleic acid sequences are contained in a vector. 39. The method of any one of claims 37 to 39. (Item 41) 342. The method of claim 341, wherein the vector is a retroviral vector. (Item 42) A composition comprising: a) a variant of human c-Kit that comprises an activating mutation; and b) an antigen-recognizing receptor that binds to an antigen. (Item 43) 43. The composition of claim 42, wherein the c-Kit mutant is operably linked to a first promoter. (Item 44) 44. The composition of claim 42 or 43, wherein the antigen-recognizing receptor is operably linked to a second promoter. (Item 45) 45. The composition of claim 43 or 44, wherein one or both of the first and second promoters are inducible promoters. (Item 46) 46. ​​The composition of item 45, wherein the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter. (Item 47) A nucleic acid composition comprising: (a) a first polynucleotide encoding an antigen-recognizing receptor; and (b) a second polynucleotide encoding a variant of human c-Kit that contains an activating mutation. (Item 48) 48. The nucleic acid composition of item 47, further comprising a first promoter operably linked to the c-Kit mutant. (Item 49) 49. The nucleic acid composition of item 47 or 48, further comprising a second promoter operably linked to the antigen-recognizing receptor. (Item 50) 50. The nucleic acid composition of item 48 or 49, wherein one or both of the first and second promoters are inducible promoters. (Item 51) 51. The nucleic acid composition of item 50, wherein the inducible promoter is selected from the group consisting of an NFAT transcription response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter. (Item 52) 52. The nucleic acid composition of any one of items 47 to 51, wherein one or both of the first and second polynucleotides are contained in a vector. (Item 53) 53. The nucleic acid composition of item 52, wherein the vector is a retroviral vector. (Item 54) 54. A vector comprising the nucleic acid composition of any one of items 47 to 53. (Item 55) A cell comprising the composition of any one of items 42 to 46, or the nucleic acid composition of any one of items 47 to 53, or the vector of item 54. (Item 56) 56. A pharmaceutical composition comprising the cells of any one of items 1 to 36 and 55 and a pharmaceutically acceptable excipient. (Item 57) 57. The pharmaceutical composition according to item 56, further comprising an inhibitor of c-Kit. (Item 58) 58. The pharmaceutical composition of item 57, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof. (Item 59) 59. A pharmaceutical composition according to any one of items 56 to 58 for treating and / or preventing a neoplasm, a pathogen infection or an infectious disease. (Item 60) 60. A method of reducing tumor burden in a subject, comprising administering to the subject a cell according to any one of items 1 to 36 and 55 or a pharmaceutical composition according to any one of items 56 to 59. (Item 61) 61. The method of claim 60, wherein the number of tumor cells is reduced, tumor size is reduced, and / or the tumor is eradicated in the subject. (Item 62) 60. A method for treating and / or preventing a neoplasm, comprising administering to said subject a cell according to any one of items 1 to 36 and 55 or a pharmaceutical composition according to any one of items 56 to 59. (Item 63) 60. A method of extending the survival time of a subject having a neoplasm, comprising administering to said subject a cell according to any one of items 1 to 36 and 55 or a pharmaceutical composition according to any one of items 56 to 59. (Item 64) 64. The method of any one of items 60 to 63, wherein the tumor or neoplasm is a solid tumor. (Item 65) Item 65. The method of item 64, wherein the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, cholangiocarcinoma, and combinations thereof. (Item 66) 66. The method of item 64 or 65, wherein the solid tumor is mesothelioma. (Item 67) 66. The method of item 64 or 65, wherein the solid tumor is lung cancer. (Item 68) 68. The method of any one of items 50 to 67, further comprising administering an inhibitor of c-Kit. (Item 69) 69. The method of item 68, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof. (Item 70) A kit comprising the cell of any one of items 1 to 36 and 55, the composition of any one of items 42 to 46, the nucleic acid composition of any one of items 47 to 53, or the vector of item 54. (Item 71) 71. The kit of item 70, further comprising written instructions for treating and / or preventing a neoplasm, pathogen infection, or infectious disease.

[0030] 4. Brief description of the drawings The following detailed description, given by way of example but not intended to limit the subject matter of the present disclosure to the particular embodiments described, can be understood in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0031] [Figure 1]1A and 1B show compositions according to certain embodiments of the presently disclosed subject matter. The composition shown in FIG. 1A, i.e., "M28z-KITv" (also referred to as "M28z-KITm"), comprises c-Kit mutant D816V and a second-generation CAR comprising an anti-mesothelin (MSLN) scFv, a CD28 transmembrane domain, a CD28 cytoplasmic signaling domain, and a CD3 zeta signaling domain. The composition shown in FIG. 1B, i.e., "Mz-KITv," comprises c-Kit mutant D816V and a first-generation CAR comprising an anti-mesothelin (MSLN) scFv, a CD28 transmembrane domain, and a CD3 zeta signaling domain. LTR indicates long terminal repeat.

[0032] [Figure 2-1] FIG. 2 shows the transduction efficiency of various constructs into T cells. [Figure 2-2] FIG. 2 shows the transduction efficiency of various constructs into T cells.

[0033] [Figure 3] Figures 3A and 3B show that M28z-KITv CAR-Ts exhibited constitutively activated pKIT signaling. Figure 3A shows Western blot results showing that M28z-KITv CAR-Ts exhibited p-KIT activity without SCF, but M28z did not express KIT protein. Figure 3B shows that M28z-KITv CAR-Ts exhibited higher p-STAT3 and p-STAT5 activity than the M28z-KITwt control.

[0034] [Figure 4] Figure 4 shows the cumulative expansion of CAR-T cells during continuous co-culture. Arrows indicate the time points of T cell restimulation (E:T=3:1) with A549GM tumor cells.

[0035] [Figure 5]Figure 5 shows the enhanced proliferation of M28z CAR T cells and M28z-KITm CAR T cells. Far red cell trace 7 days after the first antigen stimulation (E:T=2:1). Target cells were A549GM cells.

[0036] [Figure 6] Figure 6 shows the proliferation of M28z CAR T cells and M28z-KITm CAR T cells. Far red cell trace. Day 7 after the first antigen stimulation (E:T=2:1). Target cells were A549GM cells.

[0037] [Figure 7] Figures 7A and 7B show the cytolytic activity of Mz CAR T cells, M28z CAR T cells, P28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells in donor 1. Figure 7A shows the cytolytic activity at 4 hours. Figure 7B shows the cytolytic activity at 18 hours. Target cells were high MLSN A549M cells.

[0038] [Figure 8] Figures 8A and 8B show the cytolytic activity of Mz CAR T cells, M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells in donor 2 after two stimulations (E:T=3:1) with target cells (high MLSN A549GM cells) every four days. Figure 8A shows the cytolytic activity 4 hours after the second antigen stimulation. Figure 8B shows the cytolytic activity 18 hours after the second antigen stimulation.

[0039] [Figure 9] Figure 9 shows the cytolytic activity of Mz, M28z, Mz-KITv, or M28z-KITv CAR T cells. Target cells were MSLN low A549G cells.

[0040] [Figure 10]Figures 10A and 10B show PD1 expression in M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells after antigen stimulation with A549GM cells. Figure 10A shows CD4+ T cells. Figure 10B shows CD8+ T cells.

[0041] [Figure 11-1] Figures 11A and 11B show the KITv CAR T cell phenotype after antigen stimulation. Figure 11A shows the expression of stem cell-like memory T cells (TSCM) cells of M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells after stimulation with A546GM cells every 4 days (E:T=3:1). Figure 11B shows the released IFN-γ, TNF-α, and IL-2 assessed by Luminex assay after 18 hours of co-culture of CAR T cells with MSLN+ cells (E:T=3:1). [Figure 11-2] Figures 11A and 11B show the KITv CAR T cell phenotype after antigen stimulation. Figure 11A shows the expression of stem cell-like memory T cells (TSCM) cells of M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells after stimulation with A546GM cells every 4 days (E:T=3:1). Figure 11B shows the released IFN-γ, TNF-α, and IL-2 assessed by Luminex assay after 18 hours of co-culture of CAR T cells with MSLN+ cells (E:T=3:1).

[0042] [Figure 12-1]Figures 12A-12D show the in vivo efficacy of M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells against MSLN-low lung tumors. Mice bearing established MSLN-low A549G lung tumors were treated with a single dose of 1 x 10 T cells containing M28z, Mz-KITv, or M28z-KITv. UT indicates the untreated control. Figure 12A shows the results for UT. Figure 12B shows the results for M28z CAR T cells. Figure 12C shows the results for M28z-KITv CAR T cells. Figure 12D shows the results for Mz-KITv CAR T cells. [Figure 12-2] Figures 12A-12D show the in vivo efficacy of M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells against MSLN-low lung tumors. Mice bearing established MSLN-low A549G lung tumors were treated with a single dose of 1 x 10 T cells containing M28z, Mz-KITv, or M28z-KITv. UT indicates the untreated control. Figure 12A shows the results for UT. Figure 12B shows the results for M28z CAR T cells. Figure 12C shows the results for M28z-KITv CAR T cells. Figure 12D shows the results for Mz-KITv CAR T cells.

[0043] [Figure 13-1] Figures 13A-13D show the in vivo efficacy of T cells containing M28z, Mz-KITv, or M28z-KITv against MSLN-high lung tumors. Mice bearing established MSLN-high A549GM lung tumors were treated with a single dose of 1 x 10 T cells containing M28z, Mz-KITv, or M28z-KITv. UT indicates the untreated control. Figure 13A shows the results for UT. Figure 13B shows the results for M28z CAR T cells. Figure 13C shows the results for M28z-KITv CAR T cells. Figure 13D shows the results for Mz-KITv CAR T cells. [Figure 13-2]Figures 13A-13D show the in vivo efficacy of T cells containing M28z, Mz-KITv, or M28z-KITv against MSLN-high lung tumors. Mice bearing established MSLN-high A549GM lung tumors were treated with a single dose of 1 x 10 T cells containing M28z, Mz-KITv, or M28z-KITv. UT indicates the untreated control. Figure 13A shows the results for UT. Figure 13B shows the results for M28z CAR T cells. Figure 13C shows the results for M28z-KITv CAR T cells. Figure 13D shows the results for Mz-KITv CAR T cells.

[0044] [Figure 14-1] Figures 14A-14C show Kaplan-Meier survival curves for the in vivo treated mice shown in Figures 12A-12D and 13A-13D. Figure 14A shows the survival curve for mice bearing established low-antigen (mesothelin)-expressing lung tumors. Figure 14B shows the survival curve for mice bearing established high-antigen (mesothelin)-expressing lung tumors. Figure 14C shows FACS measurements of mesothelin expression levels in low-mesothelin-expressing lung cancer (A549G) and high-mesothelin-expressing lung cancer (A549GM). [Figure 14-2] Figures 14A-14C show Kaplan-Meier survival curves for the in vivo treated mice shown in Figures 12A-12D and 13A-13D. Figure 14A shows the survival curve for mice bearing established low-antigen (mesothelin)-expressing lung tumors. Figure 14B shows the survival curve for mice bearing established high-antigen (mesothelin)-expressing lung tumors. Figure 14C shows FACS measurements of mesothelin expression levels in low-mesothelin-expressing lung cancer (A549G) and high-mesothelin-expressing lung cancer (A549GM).

[0045] [Figure 15] Figure 15 shows the sensitivity of M28z and M28z-KITv CAR T cells to clinical tyrosine kinase inhibitors.

[0046] [Figure 16-1] Figures 16A-16C show the antitumor activity of M28z, Mz-KITv, or M28z-KITv CAR T cells against MSLN-high lung tumors. NSG mice bearing established MSLN-high A549GM lung tumors were treated with a single dose of 1 x 10 M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: untreated control. Figure 16A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 16B and 16C show Kaplan-Meier survival analysis of mice demonstrating the in vivo efficacy of iv administration of different CAR T cells. *, P<0.05. ***, P<0.001. [Figure 16-2] Figures 16A-16C show the antitumor activity of M28z, Mz-KITv, or M28z-KITv CAR T cells against MSLN-high lung tumors. NSG mice bearing established MSLN-high A549GM lung tumors were treated with a single dose of 1 x 10 M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: untreated control. Figure 16A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 16B and 16C show Kaplan-Meier survival analysis of mice demonstrating the in vivo efficacy of iv administration of different CAR T cells. *, P<0.05. ***, P<0.001. [Figure 16-3] Figures 16A-16C show the antitumor activity of M28z, Mz-KITv, or M28z-KITv CAR T cells against MSLN-high lung tumors. NSG mice bearing established MSLN-high A549GM lung tumors were treated with a single dose of 1 x 10 M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: untreated control. Figure 16A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 16B and 16C show Kaplan-Meier survival analysis of mice demonstrating the in vivo efficacy of iv administration of different CAR T cells. *, P<0.05. ***, P<0.001.

[0047] [Figure 17-1] Figures 17A-17C show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing lung tumors. NSG mice bearing established low-MSLN A549G lung tumors were treated with a single dose of 1 x 105 M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: untreated control. Figure 17A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 17B and 17C show Kaplan-Meier survival analysis of mice demonstrating the in vivo efficacy of iv administration of different CAR T cells. **, P<0.01. [Figure 17-2] Figures 17A-17C show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing lung tumors. NSG mice bearing established low-MSLN A549G lung tumors were treated with a single dose of 1 x 105 M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: untreated control. Figure 17A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 17B and 17C show Kaplan-Meier survival analysis of mice demonstrating the in vivo efficacy of iv administration of different CAR T cells. **, P<0.01. [Figure 17-3] Figures 17A-17C show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing lung tumors. NSG mice bearing established low-MSLN A549G lung tumors were treated with a single dose of 1 x 105 M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: untreated control. Figure 17A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 17B and 17C show Kaplan-Meier survival analysis of mice demonstrating the in vivo efficacy of iv administration of different CAR T cells. **, P<0.01.

[0048] [Figure 18-1] Figures 18A and 18B show Kaplan-Meier survival analyses comparing the in vivo efficacy of intrapleural administration of M28z, Mz-KITv, or M28z-KITv CAR T cells in a pleural mesothelioma tumor model. NSG mice bearing established MSLN-high MGM mesothelioma were treated with a single dose of 5 x 104 P28z, Mz, M28z, M28z-KITv, and Mz-KITv CAR T cells. *, P<0.05. Figure 18A shows the comparison among all groups. Figure 18B shows the comparison between the M28z and M28z-KITv groups and between the Mz and Mz-KITv groups. [Figure 18-2] Figures 18A and 18B show Kaplan-Meier survival analyses comparing the in vivo efficacy of intrapleural administration of M28z, Mz-KITv, or M28z-KITv CAR T cells in a pleural mesothelioma tumor model. NSG mice bearing established MSLN-high MGM mesothelioma were treated with a single dose of 5 x 104 P28z, Mz, M28z, M28z-KITv, and Mz-KITv CAR T cells. *, P<0.05. Figure 18A shows the comparison among all groups. Figure 18B shows the comparison between the M28z and M28z-KITv groups and between the Mz and Mz-KITv groups.

[0049] [Figure 19-1] Figures 19A and 19B show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing mesothelioma. Figure 19A shows low or high MSLN protein expression in MSTO cells to generate MG-LM and MGM cells, respectively. Figure 19B shows NSG mice bearing established low-MSLN mesothelioma (MG-LM) were treated with a single dose of 5 x 10 P28z, M28z, M28z-KITv, or Mz-KITv CAR T cells. Kaplan-Meier survival analysis compared the in vivo efficacy of intrapleural administration of different CAR T cells. *, P<0.05. **, P<0.01. [Figure 19-2]Figures 19A and 19B show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing mesothelioma. Figure 19A shows low or high MSLN protein expression in MSTO cells to generate MG-LM and MGM cells, respectively. Figure 19B shows NSG mice bearing established low-MSLN mesothelioma (MG-LM) were treated with a single dose of 5 x 10 P28z, M28z, M28z-KITv, or Mz-KITv CAR T cells. Kaplan-Meier survival analysis compared the in vivo efficacy of intrapleural administration of different CAR T cells. *, P<0.05. **, P<0.01.

[0050] [Figure 20-1] Figure 20 shows the p-ERK signal of CAR T cells after antigen stimulation. After co-culture with MGM cells for 5 minutes (E:T=1:2), the p-ERK levels of CD4+ and CD8+ CAR T cells were measured by FACS. Both CD4 and CD8 of M28z-KITv and Mz-KITv CAR T cells had stronger p-ERK activity than M28z. [Figure 20-2] Figure 20 shows the p-ERK signal of CAR T cells after antigen stimulation. After co-culture with MGM cells for 5 minutes (E:T=1:2), the p-ERK levels of CD4+ and CD8+ CAR T cells were measured by FACS. Both CD4 and CD8 of M28z-KITv and Mz-KITv CAR T cells had stronger p-ERK activity than M28z. [Figure 20-3] Figure 20 shows the p-ERK signal of CAR T cells after antigen stimulation. After co-culture with MGM cells for 5 minutes (E:T=1:2), the p-ERK levels of CD4+ and CD8+ CAR T cells were measured by FACS. Both CD4 and CD8 of M28z-KITv and Mz-KITv CAR T cells had stronger p-ERK activity than M28z.

[0051] [Figure 21-1]Figures 21A and 21B show that CAR T cells obtained from mice were exposed to high mesothelin-expressing mesothelioma cells and analyzed for PD1 expression. Figure 21A shows quantification of PD1 expression at different E:T ratios. Figure 21B shows a flow cytometry graph measuring PD1 expression at different E:T ratios. [Figure 21-2] Figures 21A and 21B show that CAR T cells obtained from mice were exposed to high mesothelin-expressing mesothelioma cells and analyzed for PD1 expression. Figure 21A shows quantification of PD1 expression at different E:T ratios. Figure 21B shows a flow cytometry graph measuring PD1 expression at different E:T ratios.

[0052] [Figure 22-1] Figures 22A and 22B show enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells. After 24 hours of co-culture with MSLN+ tumor cells, M28z and M28z-KITv CD8 CAR T cells were collected for analysis by nanostring for CAR T panel genes, n=3 for each group. Figure 22A shows that 87 of 780 detected genes had significant fold changes. Figure 22B shows a heatmap of pathway scores showing enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells. Figure 22C provides upregulated gene pathways in KIT CAR T cells. [Figure 22-2]Figures 22A and 22B show enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells. After 24 hours of co-culture with MSLN+ tumor cells, M28z and M28z-KITv CD8 CAR T cells were collected for analysis by nanostring for CAR T panel genes, n=3 for each group. Figure 22A shows that 87 of 780 detected genes had significant fold changes. Figure 22B shows a heatmap of pathway scores showing enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells. Figure 22C provides upregulated gene pathways in KIT CAR T cells. [Figure 22-3] Figures 22A and 22B show enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells. After 24 hours of co-culture with MSLN+ tumor cells, M28z and M28z-KITv CD8 CAR T cells were collected for analysis by nanostring for CAR T panel genes, n=3 for each group. Figure 22A shows that 87 of 780 detected genes had significant fold changes. Figure 22B shows a heatmap of pathway scores showing enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells. Figure 22C provides upregulated gene pathways in KIT CAR T cells.

[0053] [Figure 23-1]Figures 23A-23B show significant upregulation of interferon signaling genes in M28z-KITv CAR T cells compared to M28z CAR T cells. After 24 hours of co-culture with MSLN+ cancer cells, M28z and M28z-KITv CD8 CAR T cells were collected for analysis by nanostring for CAR T panel genes, n=3 for each group. Expression of type I interferon signaling genes (Figure 23A) and type II interferon signaling genes (Figure 23B) was significantly increased in M28z-KITv CAR T cells. [Figure 23-2] Figures 23A-23B show significant upregulation of interferon signaling genes in M28z-KITv CAR T cells compared to M28z CAR T cells. After 24 hours of co-culture with MSLN+ cancer cells, M28z and M28z-KITv CD8 CAR T cells were collected for analysis by nanostring for CAR T panel genes, n=3 for each group. Expression of type I interferon signaling genes (Figure 23A) and type II interferon signaling genes (Figure 23B) was significantly increased in M28z-KITv CAR T cells. DETAILED DESCRIPTION OF THE INVENTION

[0054] 5. Detailed Description of the Invention The presently disclosed subject matter provides cells comprising a c-Kit mutant, where the c-Kit mutant comprises an activating mutation. The cells may be genetically modified immunoresponsive cells (e.g., T cells or NK cells), and the cells comprise an antigen-recognition receptor (e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR)). The presently disclosed subject matter also provides methods of using such cells to induce and / or enhance an immune response to a target antigen and / or to treat and / or prevent neoplasms, pathogen infections, or other diseases / disorders (e.g., diseases / disorders in which an increase in antigen-specific immune response is desired). The presently disclosed subject matter is based, at least in part, on the discovery that a c-Kit mutant (e.g., c-KitD816V) can enhance cell proliferation of cells (e.g., T cells) comprising an antigen-recognition receptor (e.g., a CAR).

[0055] Non-limiting embodiments of the present disclosure are described herein and by way of example.

[0056] For clarity of the disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definition; 5.2.c-Kit mutant; 5.3.Cell; 5.4. Antigen recognition receptors; 5.5. Dominant Negative Programmed Death 1 (PD-1 DN) 5.6. Compositions and Vectors; 5.7. Polypeptides and analogs; 5.8.Administration; 5.9. Preparations; 5.10. Treatment methods; and 5.11.Kit

[0057] 5.1.Definition Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The following references provide those skilled in the art with general definitions of many of the terms used in the subject matter of this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0058] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, per the practice of the art. Alternatively, "about" can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, e.g., within 5-fold or within 2-fold.

[0059] By "immunoresponsive cell" is meant a cell or precursor, or progeny thereof, that functions in an immune response.

[0060] "Activating immunoresponsive cells" refers to the induction of signal transduction or changes in protein expression in cells that result in the initiation of an immune response. For example, when CD3 chains form clusters in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade occurs. In certain embodiments, when an endogenous TCR or exogenous CAR binds to an antigen, an immune synapse is formed, involving the clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the bound receptor. This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates a T cell activation pathway that ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors induce general gene expression in T cells, increasing IL-2 production for proliferation and expression of master regulator T cell proteins to initiate a T cell-mediated immune response.

[0061] "Stimulating immunoresponsive cells" refers to signals that result in a strong and sustained immune response. In various embodiments, this occurs after immune cell (e.g., T cell) activation or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Administering multiple stimulatory signals can be important for initiating a strong and long-lasting T cell-mediated immune response. T cells can be quickly inhibited and unable to respond to antigens. While the effects of these costimulatory signals can vary, they generally result in increased gene expression, generating long-lived, proliferative, and anti-apoptotic T cells that respond strongly to antigens for complete and sustained eradication.

[0062] The term "antigen-recognizing receptor," as used herein, refers to a receptor that is capable of activating an immune or immunoresponsive cell (e.g., a T cell) in response to binding to an antigen.

[0063] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fe fragment of intact antibodies, clear more rapidly from the circulation and may have less nonspecific tissue binding than intact antibodies (Wahl et al., J Nucl Med (1983); 24:316-325). As used herein, antibodies include natural whole antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain variable fragments (scFv), fusion polypeptides, and non-traditional antibodies. In certain embodiments, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H ) and heavy chain constant region (C H The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (referred to herein as V L ) and light chain constant C L The light chain constant region consists of one domain, C L It consists of: V H Area and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0064] As used herein, "CDR" is defined as the complementarity-determining region amino acid sequence of an antibody, which is the hypervariable region of an immunoglobulin heavy chain and light chain. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for binding of the antibody to an antigen or epitope. In certain embodiments, the CDR region is delineated using the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services (1991); NIH Publication No. 91-3242).

[0065] As used herein, the term "single-chain variable fragment" or "scFv" refers to a V H ::V L Covalently linked immunoglobulin heavy chains (V) to form heterodimers H ) and light chain (V L ) is a fusion protein of the variable region of V H and V Lare either directly joined or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), thereby forming V H The N-terminus of L or V H The C-terminus of L The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility. "Linker," as used herein, is intended to mean a functional group (e.g., a chemical or polypeptide) that covalently joins two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a "peptide linker" is a peptide linker that is used to link two proteins together (e.g., V H Domains and V L In certain embodiments, the linker comprises the sequence shown in SEQ ID NO: 16, provided below: GGGGSGGGGSGGGGS [SEQ ID NO: 16]

[0066] In one particular embodiment, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is set forth in SEQ ID NO: 17, provided below: GGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCA [SEQ ID NO: 17]

[0067] In one particular embodiment, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is set forth in SEQ ID NO: 18, provided below. GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCA [SEQ ID NO: 18]

[0068] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are characterized by the VFv polypeptide described by Huston et al., Proc. Nat. Acad. Sci. USA (1988); 85:5879-5883. H and V L The coding sequence may be expressed from a nucleic acid containing the coding sequence, see U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle(2013); 4(1):79-86; Shieh et al., J Imunol(2009);183(4):2277-85; Giomarelli et al., Thromb Haemost(2007);97(6):955-63; Fife et al., JCI(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 (Peter et al., J Biol Chem (2003); 25278(38):36740-7; Xie et al., Nat Biotech (1997); 15(8):768-71; Ledbetter et al., Crit Rev Immunol (1997); 17(5-6):427-55; Ho et al., BioChem Biophys Acta (2003); 1638(3):257-66).

[0069] As used herein, the term "affinity" refers to a measure of binding strength. Affinity may depend on the closeness of the stereochemical fit between the antibody combining site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "avidity," which refers to the strength of antigen-antibody binding after reversible complex formation. Methods for calculating the affinity of an antibody for an antigen are known in the art and include, but are not limited to, various antigen binding experiments, such as functional assays (e.g., flow cytometry assays).

[0070] The term "chimeric antigen receptor" or "CAR," as used herein, refers to a molecule comprising an intracellular signaling domain capable of activating or stimulating immune response cells, and an extracellular antigen-binding domain fused to a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be derived by fusing to the variable heavy and light chain regions of an antibody. Alternatively, or in addition, the scFv can be derived from a Fab (e.g., obtained from a Fab library, instead of being derived from an antibody). In certain embodiments, the scFv is fused to a transmembrane domain and then fused to the intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for the antigen.

[0071] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule that encodes a polypeptide of interest. Such nucleic acid molecules need not be 100% homologous or identical to an endogenous nucleic acid sequence, but may exhibit substantial identity.

[0072] A polynucleotide having "substantial identity" or "substantial homology" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to the pairing of complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringency conditions. (Wahl et al., Methods Enzymol. (1987); 152:399; Kimmel, Methods Enzymol. (1987); 152:507).

[0073] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than about 75 mM trisodium citrate, such as less than about 500 mM NaCl and less than about 50 mM trisodium citrate, or less than about 250 mM NaCl and less than about 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, for example, at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, detergent concentration, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, is well known to those skilled in the art. Varying levels of stringency can be achieved by combining these various conditions as needed. In certain embodiments, hybridization is performed at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization is performed at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization is performed at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be readily apparent to those of skill in the art.

[0074] In most applications, the washing step after hybridization also varies in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, a stringent salt concentration for the washing step can be less than about 30 mM NaCl and less than about 3 mM trisodium citrate, such as less than about 15 mM NaCl and less than about 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step usually include a temperature of at least about 25°C, at least about 42°C, or at least about 68°C. In certain embodiments, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash steps are performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to one of skill in the art. Hybridization techniques are well known to those of skill in the art and have been described (Benton et al., Science (1977); 196:180; Grunstein et al., Proc. Natl. Acad. Sci., USA (1975); 72:3961; Ausubel et al., Current Protocols in Molecular Biology (2001); Wiley Interscience, New York; Berger et al., Guide to Molecular Cloning Techniques (1987); Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, (1987); Cold Spring Harbor Laboratory Press, New York).

[0075] "Substantially identical" or "substantially homologous" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid or nucleic acid sequence used for comparison.

[0076] Sequence identity can be determined using sequence analysis software (e.g., Sequence Analysis The degree of identity can be determined by using the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, with a probability score between e-3 and e-100 indicating closely related sequences.

[0077] "Analog" means a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0078] The term "ligand," as used herein, refers to a molecule that binds to a receptor. In certain embodiments, a ligand binds to a receptor on another cell, allowing recognition and / or interaction between the cells.

[0079] The term "constitutive expression" or "constitutively expressed" as used herein refers to expression under or being expressed under all physiological conditions.

[0080] By "disease" is meant any condition, disease or disorder that damages or interferes with the normal function of a cell, tissue or organ, for example, neoplasms and pathogenic infection of cells.

[0081] "Effective amount" means an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to stop, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0082] "Enhancing tolerance" means preventing the activity of autoreactive or immunoresponsive cells that target the transplanted organ or tissue.

[0083] "Endogenous" means a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0084] "Exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as a foreign, heterologous, or overexpressed nucleic acid molecule or polypeptide. "Exogenous" nucleic acid refers to a nucleic acid that is not present in a natural, wild-type cell; for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or a different sequence compared to its natural endogenous counterpart, may be introduced into the cell itself or its precursor by genetic engineering, and may, if necessary, be linked to an alternative control sequence, such as a non-native promoter or secretory sequence.

[0085] By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism, or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0086] "Modulate" means to alter, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0087] By "increase" is meant to positively alter by at least about 5%. The alteration may be up to about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0088] By "reducing" is meant to negatively alter by at least about 5%. The alteration may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even up to about 100%.

[0089] The terms "isolated," "purified," or "biologically pure" refer to material that is free, to varying degrees, from components that normally accompany it as found in its natural state. "Isolate" refers to a degree of separation from the original source or environment. "Purify" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is substantially free from other substances, such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified if it is produced by recombinant DNA techniques, but is substantially free from cellular material, viral material, and culture medium, or if chemically synthesized, from chemical precursors and other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that may be subject to modifications, such as phosphorylation or glycosylation, different modifications may occur in different isolated proteins, which may be purified separately.

[0090] By "isolated cell" is meant a cell that has been separated from the molecules and / or cellular components that naturally accompany the cell.

[0091] The term "antigen-binding domain," as used herein, refers to a domain capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0092] "Neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration to or invasion of other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell multiplication. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, organs selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or tissues or cell types thereof. Neoplasms include cancers, such as sarcoma, carcinoma, or plasmacytoma (a malignant tumor of plasma cells). In certain embodiments, the neoplasm is cancer. In certain embodiments, the neoplasm is a solid tumor.

[0093] By "receptor" is meant a polypeptide or portion thereof present on a cell membrane that selectively binds one or more ligands.

[0094] "Recognize" means selectively binding to a target. T cells that recognize tumors can express receptors (e.g., TCRs or CARs) that bind to tumor antigens.

[0095] "Reference" or "control" refers to a standard for comparison. For example, the level of scFv antigen binding by cells expressing a CAR and an scFv can be compared to the level of scFv antigen binding in corresponding cells expressing a CAR alone.

[0096] By "secreted" is meant a polypeptide that is released from the cell by the secretory pathway via the endoplasmic reticulum, the Golgi apparatus, and as vesicles that transiently fuse with the cell plasma membrane, releasing the protein outside the cell.

[0097] "Signal sequence" or "leader sequence" means a peptide sequence (eg, 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs entry into the secretory pathway. Exemplary leader sequences include, but are not limited to, the IL-2 signal sequence: MYRMQLLSCIALSLALVTNS [SEQ ID NO:43] (human), MYSMQLASCVTLTLVLLVNS [SEQ ID NO:44] (mouse); kappa leader sequence: METPAQLLFLLLLWLPDTTG [SEQ ID NO:45] (human), METDTLLLWVLLLWVPGSTG [SEQ ID NO:46] (mouse); CD8 leader sequence: MALPVTALLLPLALLLHAARP [SEQ ID NO:47] (human); truncated human CD8 signal peptide: MALPVTALLLPLALLLHA [SEQ ID NO:48] (human); albumin signal sequence: MKWVTFISLLFSSAYS [SEQ ID NO:49] (human); and prolactin signal sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO:20] (human). "Soluble" refers to a polypeptide that is freely diffusible in an aqueous environment (e.g., not membrane-bound).

[0098] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds to a biological molecule (e.g., a polypeptide) of interest, but does not substantially recognize or substantially bind to other molecules in a sample, e.g., a biological sample, that naturally contains a polypeptide of the present disclosure.

[0099] The terms "comprises," "comprising," and "comprising" are intended to have the broad meaning given them in U.S. patent law and may mean "includes," "including," etc.

[0100] As used herein, "treatment" refers to a therapeutic intervention that attempts to alter the disease course of the treated individual or cell, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improving prognosis. By preventing the progression of a disease or disorder, treatment can prevent the deterioration of the disorder in a subject who has been affected or diagnosed, or a subject suspected of having the disorder, but treatment can also prevent the onset of the disorder or the symptoms of the disorder in a subject who is at risk for the disorder or suspected of having the disorder.

[0101] An "individual" or "subject," as used herein, refers to a vertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, game animals, rodents, and pet animals. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys. The term "immunocompromised," as used herein, refers to a subject with an immune deficiency. Subjects are highly susceptible to opportunistic infections, which are infections caused by organisms that do not normally cause disease in people with healthy immune systems but can affect people with poorly functioning or suppressed immune systems.

[0102] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter.

[0103] 5.2.c-Kit mutants The proto-oncogene KIT is a receptor tyrosine kinase protein and is also known as CD117; KIT; PBT; stem cell growth factor receptor (SCFR); MASTC. GenBank ID: 3815 (human), 16590 (mouse). Protein products of KIT include, but are not limited to, NCBI reference sequences NP_000213 (human isoform 1), NP 001122733 (mouse isoform 1).

[0104] c-Kit, also known as CD117, is a cytokine receptor expressed on the surface of hematopoietic stem cells as well as other cell types. Signaling through c-Kit plays a role in cell survival, proliferation, and differentiation (Ceredig et al., Nat Rev Immunol (2002); 2(11):888-97).

[0105] c-Kit binds to stem cell factor (SCF). Upon binding, c-Kit and SCF form a dimer, which activates its intrinsic tyrosine kinase activity and then phosphorylates and activates signaling molecules that propagate signals in the cell. The c-Kit activating mutations of the present disclosure (e.g., the D816V mutation) result in constitutive activation without SCF, e.g., without forming a cKit / SCF dimer (Hirota, et al., Science (1998);279(5350):577-80;Kitamura et al., Mut Res (2001):165-71).

[0106] In certain embodiments, the c-Kit mutant is a human c-Kit mutant. In certain embodiments, the human c-Kit protein comprises or consists of a sequence having NCBI reference number NP_000213 (SEQ ID NO: 1). SEQ ID NO: 1 is provided below. [ka]

[0107] The subject cells of the present disclosure comprise a c-Kit mutant. In certain embodiments, the c-Kit mutant comprises an activating mutation.

[0108] In certain embodiments, an activating mutation is a gain-of-function mutation, hi certain embodiments, an activating mutation is a mutation whose gene product has an enhanced effect compared to a gene product that does not have such a mutation (e.g., a wild-type protein).

[0109] In certain embodiments, the activating mutation (e.g., D816V mutation) is present in early lineage hematopoietic cells and is lost during maturation. In certain embodiments, the activating mutation of c-Kit results in c-Kit activation independent of c-Kit's interaction with its ligand (e.g., SCF). In certain embodiments, the activating mutation of c-Kit results in c-Kit activation without a c-Kit ligand, e.g., SCF. In certain embodiments, the activating mutation of c-Kit results in constitutive activation of c-Kit. In certain embodiments, the activating mutation of c-Kit results in c-Kit activation independent of inhibition of tyrosine phosphatase-1 (SHP-1) and / or tyrosine phosphatase-2 (SHP-2). In certain embodiments, the activating mutation of c-Kit results in c-Kit activation in the presence of inhibition of SHP-1 and / or SHP-2.

[0110] Activating c-Kit mutations promote proliferation and anti-apoptosis in cells containing mutant c-Kit, and confers resistance to PD-L1 / 2-PD-1 inhibition in these cells.

[0111] In certain embodiments, the activating mutation is located within the intracellular region of human cKIT, e.g., human cKIT consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the intracellular region of human cKIT comprises amino acids 544-977 of SEQ ID NO: 1. In certain embodiments, the activating mutation is located within amino acids 816-826 of human cKIT, e.g., human cKIT consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the activating mutation is located at amino acid position 816 or amino acid position 822. In certain embodiments, the activating mutation is located within amino acids 550-570 of human cKIT, e.g., human cKIT consisting of the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the activating mutation is located at amino acid position 560. Non-limiting examples of c-Kit activating mutations include D816V, D816Y, D816H, D816F, N822K, V560G, or a combination thereof. In certain embodiments, the activating mutation is D816V.

[0112] The c-Kit mutant may be operably linked to a promoter. The promoter may be endogenous or exogenous. Non-limiting examples of exogenous promoters include the elongation factor (EF)-1 promoter, the cytomegalovirus immediate early promoter (CMV) promoter, the simian virus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, and the metallothionein promoter. In certain embodiments, the promoter is an inducible promoter. Non-limiting examples of inducible promoters are selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter. The inducible promoter can control the activation of c-Kit; for example, by controlling the inducible promoter, c-Kit is activated only upon activation of cells (e.g., T cells or CAR-T cells) containing the c-Kit mutant.

[0113] In certain embodiments, the c-Kit variant comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 2, or a portion thereof. In certain embodiments, the c-Kit variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, or a portion thereof. In certain embodiments, the c-Kit variant comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:2 that is at least 50, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350, or at least 400, or at least 450, or at least 500, or at least 550, or at least 600, or at least 650, or at least 700, or at least 750, or at least 800, or at least 850, or at least 900, or at least 950, and up to 976 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the c-Kit variant comprises or consists of the amino acid sequence of amino acids 1-976, 1-200, 400-976, 500-976, or 543-976 of SEQ ID NO:2. In certain embodiments, the c-Kit variant comprises or consists of amino acids 543-976 of SEQ ID NO:2.

[0114] SEQ ID NO:2 is provided below. [ka] [ka]

[0115] 5.3.Cells The presently disclosed subject matter provides cells comprising a c-Kit mutant disclosed herein (e.g., as disclosed in Section 5.2). In certain embodiments, the c-Kit mutant is an exogenous c-Kit mutant.

[0116] In certain embodiments, the cell is selected from a cell of the lymphoid lineage and a cell of the myeloid lineage. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage.

[0117] In certain embodiments, the cell is a lymphoid lineage cell. Lymphoid lineage cells can produce antibodies, regulate the cellular immune system, detect foreign pathogens in the blood, detect cells foreign to the host, etc. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells that can differentiate into lymphoid cells. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell).

[0118] In certain embodiments, the cell is a T cell. T cells may be lymphocytes that mature in the thymus and are largely responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the presently disclosed subject matter include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells: e.g., T EM Cells and T EMRA The immunoresponsive cells can be any type of T cell, including, but not limited to, T lymphocytes (including CD4 T cells), regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by the introduction of antigen-recognition receptors, e.g., CARs or TCRs. In certain embodiments, the immunoresponsive cells are T cells. T cells are CD4+ T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + T cells.

[0119] In certain embodiments, the cell is an NK cell. Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act in innate immune responses. NK cells do not require prior activation to exert cytotoxic effects on target cells.

[0120] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, such as those described in Sadelain, M., et al. Nat Rev Cancer (2003); 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express a CAR), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising an α and β heterodimer), Panelli et al. J Immunol (2000); 164:495-504; Panelli et al., J Immunol (2000); 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont et al., Cancer Res (2005); 65:5417-5427; Papanicolaou et al., Blood (2003); 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells).

[0121] The cells (e.g., T cells) may be autologous, non-autologous (e.g., allogeneic), or derived from in vitro engineered progenitor or stem cells. In certain embodiments, the cells are allogeneic cells.

[0122] The subject cells of the present disclosure can be cells of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can differentiate.

[0123] In certain embodiments, the stem cells are pluripotent stem cells (eg, embryonic stem cells or induced pluripotent stem cells).

[0124] In certain embodiments, the cells of the present disclosure are capable of modulating the tumor microenvironment. Tumors have a microenvironment hostile to the host immune response, involving a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes infiltrating regulatory CD4 + Tumors contain various immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands targeted to immunoinhibitory receptors (CTLA-4 and PD-1) expressed by activated T cells. These mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inappropriate immune responses, but within the tumor microenvironment, these mechanisms prevent effective antitumor immune responses. Collectively, these immunosuppressive factors can induce either profound anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with targeted tumor cells.

[0125] In certain embodiments, the cells of the present disclosure have increased cell proliferation and / or cell persistence, hi certain embodiments, the immunoresponsive cells of the present disclosure have decreased apoptosis and / or anergy.

[0126] In certain embodiments, the cells further comprise an antigen-recognizing receptor (e.g., CAR or TCR) that binds to an antigen. The cells can be transduced with an exogenously activating antigen-recognizing receptor and a c-Kit mutant so that the cells co-express the antigen-recognizing receptor and the c-Kit mutant.

[0127] The c-Kit mutant may be operably linked to a first promoter. The antigen-recognizing receptor may be operably linked to a second promoter. The first promoter may be the same as the second promoter. Alternatively, the first promoter is different from the second promoter. The first and second promoters may be endogenous or exogenous. Non-limiting examples of exogenous promoters include the elongation factor (EF)-1 promoter, the cytomegalovirus immediate early promoter (CMV) promoter, the simian virus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, and the metallothionein promoter. In certain embodiments, one or both of the first and second promoters are inducible promoters. Non-limiting examples of inducible promoters are selected from the group consisting of the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter.

[0128] 5.4. Antigen Recognition Receptors In certain embodiments, the cell of the present disclosure further comprises an antigen-recognizing receptor. In certain embodiments, the antigen-recognizing receptor binds to an antigen. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen-recognizing receptor is a T cell receptor (TCR). In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule.

[0129] 5.4.1. Antigen The antigen-recognizing receptor can bind to a tumor antigen or a pathogen antigen.

[0130] In certain embodiments, the antigen-recognizing receptor binds to a tumor antigen. Any tumor antigen (antigen peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, cancer proteins. The antigen can be expressed as a peptide or as an intact protein or a portion thereof. The intact protein or a portion thereof can be natural or mutagenized. In certain embodiments, the antigen is expressed in tumor tissue. Non-limiting examples of tumor antigens include mesothelin, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-alpha, GD2, GD3, HER-2, hTERT, IL-13R-α2, K-light chain, KDR, LeY, and L1 cell grafts. tumor antigens include, but are not limited to, MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. In certain embodiments, the tumor antigen is mesothelin.

[0131] In certain embodiments, the antigen-recognizing receptor binds to mesothelin. In certain embodiments, the antigen-recognizing receptor binds to human mesothelin consisting of the sequence having NCBI reference number AAV87530.1 (SEQ ID NO: 3) or a fragment thereof. SEQ ID NO: 3 is provided below: [ka]

[0132] In certain embodiments, antigen recognition receptors for use in treating and / or preventing, e.g., pathogen infections or other infectious diseases, e.g., in immunocompromised subjects, bind to pathogen antigens. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0133] Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 (also referred to as HDLV-III, LAVE, or HTLV-III / LAV, or HIV-III); and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Fla Viridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantavirus, Bunyavirus) virus), phlebovirus, and Naira virus; Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvovirida (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (variola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), agents of non-A, non-B hepatitis (class 1 = internal infection, class 2 = parenteral infection (i.e., hepatitis C));Norwalk and related viruses and astroviruses, human papillomaviruses (i.e., HPV), JC virus, Epstein-Barr virus, Merkel cell polyomavirus);

[0134] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A streptococcus), Streptococcus agalactiae (group B streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, clostridium difficile and Actinomyces israelli.

[0135] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus. T cell receptor (TCR)

[0136] In certain embodiments, the antigen-recognition receptor is a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0137] Each chain of the TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. Each of the variable domains of both chains consists of three complementarity-determining regions (CDRs).

[0138] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247 ζ / ζ or ζ / η. When the TCR complex associates with its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0139] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the antigen-recognizing receptor is a naturally occurring TCR.

[0140] In certain embodiments, the antigen-recognizing receptor is an exogenous TCR. In certain embodiments, the antigen-recognizing receptor is a recombinant TCR. In certain embodiments, the antigen-recognizing receptor is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. Chimeric Antigen Receptors (CARs)

[0141] In certain embodiments, the antigen-recognition receptor is CAR.CAR is an engineered receptor that transfers or gives desired specificity to immune effector cells.Retroviral vectors can be used to promote the transfer of coding sequences, and CAR can be used to transfer the specificity of monoclonal antibody onto T cells.

[0142] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs provide de novo antigen recognition and bind to CD4 ζ chains via their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + T cells and CD8 +These CARs can activate both T cells and stimulate T cells. "Second-generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to T cells. "Second-generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen-recognizing receptor is a first-generation CAR. In certain embodiments, the antigen-recognizing receptor is a CAR that does not include the intracellular signaling domain of a costimulatory molecule. In certain embodiments, the antigen-recognizing receptor is a second-generation CAR.

[0143] In certain embodiments, a CAR may comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen, which may be a tumor antigen or a pathogen antigen.

[0144] 5.4.3.1. Extracellular Antigen-Binding Domain of the CAR In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen. In certain embodiments, the antigen is mesothelin. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the extracellular antigen-binding domain is an optionally cross-linked Fab. In certain embodiments, the extracellular antigen-binding domain is a F(ab)2. In certain embodiments, any of the above molecules can be included within a fusion protein containing heterologous sequences to form the extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein.

[0145] In certain non-limiting embodiments, the extracellular antigen binding domain of the CAR (e.g., embodied as an scFv or analog thereof) is about 2×10 -7 M or less dissociation constant (K d ) binds to the antigen. In certain embodiments, K d is about 2 x 10 -7 M or less, approximately 1 x 10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1 x 10 -8 M or less, approximately 9 x 10 -9 M or less, approximately 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, approximately 3 x 10 -9 or less, about 2 × 10 -9 M or less, or about 1 x 10 -9 M or less. In certain non-limiting embodiments, K d is about 3 x 10 -9 M or less. In certain non-limiting embodiments, K d is approximately 1 x 10 -9 M ~ approx. 3×10 -7 M. In certain non-limiting embodiments, K d is approximately 1.5 x 10 -9 M ~ approx. 3×10 -7 M. In certain non-limiting embodiments, K d is approximately 1.5 x 10 -9 M ~ approx. 2.7×10 -7 I am M.

[0146] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR has high binding specificity and high binding affinity for human mesothelin. For example, in such embodiments, the extracellular antigen-binding domain of the CAR (e.g., embodied as an scFv) binds to human mesothelin with an EC50 value of about 1 nM to about 25 nM as measured by enzyme-linked immunosorbent assay (ELISA). In certain embodiments, the extracellular antigen-binding domain of the CAR has an EC50 value of about 20 nM as measured by ELISA. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an anti-mesothelin antibody, or antigen-binding portion thereof, described in U.S. Patent No. 8,357,783, the entire contents of which are incorporated herein by reference. In certain embodiments, the extracellular antigen-binding domain of the CAR is derived from the heavy and light chain variable regions of an antibody that binds to human mesothelin, such as the antibody m912 disclosed in Feng et al., Mol. Cancer Therapy (2009);8(5):1113-1118, the entire contents of which are incorporated herein by reference. Antibody m912 was isolated from a human Fab library by panning against recombinant mesothelin. In certain embodiments, the extracellular antigen-binding domain of the CAR is derived from a Fab (e.g., a human or mouse Fab library).

[0147] The binding of the extracellular antigen-binding domain of the CAR (in embodiments, e.g., scFv) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the mesothelin-targeting extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, citrine, Venus, and YPet). In one embodiment, the mesothelin-targeting human scFv is labeled with GFP.

[0148] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR recognizes or binds to human mesothelin having a mesothelin level of about 1,000 or more mesothelin binding sites / cell. In certain embodiments, the extracellular antigen-binding domain of the CAR recognizes or binds to human mesothelin having a mesothelin level of about 1,000 to about 50,000 mesothelin binding sites / cell. In some embodiments, the extracellular antigen-binding domain of the CAR does not recognize or bind to human mesothelin having a mesothelin expression level of less than 1,000 mesothelin binding sites / cell, such as human mesothelin expressed in normal tissues, such as normal pleural, pericardial, and peritoneal tissues. In certain embodiments, the extracellular antigen-binding domain of the CAR does not recognize or bind to human mesothelin having a mesothelin expression level of more than 50,000 mesothelin binding sites / cell. In certain embodiments, the human scFv contained in the CAR recognizes or binds to human mesothelin having a mesothelin expression level of about 1,000 to about 50,000 mesothelin binding sites / cell. In certain embodiments, the human scFv contained in the CAR does not recognize or bind to human mesothelin having a mesothelin expression level of more than 50,000 or less than 1,000 mesothelin binding sites / cell.

[0149] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 4, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof H CDR2, and V containing the amino acid sequence shown in SEQ ID NO: 6, conservative modifications thereof H In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises a V CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:5 H CDR2, and V comprising the amino acid sequence shown in SEQ ID NO:6 HIn certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises a V CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof L In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises a V CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:8 L CDR2, and V comprising the amino acid sequence shown in SEQ ID NO:9 L Includes CDR3.

[0150] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 4, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof H CDR2, the amino acid sequence shown in SEQ ID NO: 6, including conservative modifications thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof L In certain embodiments, the extracellular antigen-binding domain comprises a V CDR3 comprising an amino acid sequence having the sequence set forth in SEQ ID NO:4. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:5 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:6 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:7 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:8 L CDR2, and V comprising the amino acid sequence shown in SEQ ID NO:9 LIn certain embodiments, the CDRs are identified according to the Kabat numbering system, including CDR3.

[0151] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises a heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 10. H In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises a light chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 11. L In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence shown in SEQ ID NO: 11 L and optionally (iii) said V H and the aforementioned V L and a linker sequence, e.g., a linker peptide, between them. In certain embodiments, the linker comprises amino acids consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V that comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 10. H For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO: 10. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V that comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 11. LFor example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO: 11. L In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 11. L In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V that comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 10. H and V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO:11. L In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence shown in SEQ ID NO: 11 L Includes:

[0152] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:10 is shown in SEQ ID NO:12.

[0153] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:11 is shown in SEQ ID NO:13.

[0154] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv) specifically binds to a human mesothelin polypeptide (e.g., a human mesothelin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3).

[0155] In certain embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 is set forth in SEQ ID NO:15.

[0156] In certain embodiments, the scFv is a human scFv. SEQ ID NOs: 4-15 are provided below: [ka] [ka]

[0157] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the mesothelin-targeting CAR of the present disclosure (e.g., the extracellular antigen-binding domain of the CAR) comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine, negatively charged amino acids include aspartic acid and glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Amino acids can also be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues within a designated sequence or CDR region are altered.

[0158] A V that has at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., SEQ ID NO: 10 or SEQ ID NO: 11). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the designated sequence(s), but retain the ability to bind to a target antigen (e.g., mesothelin). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a particular sequence (e.g., SEQ ID NO: 10 or SEQ ID NO: 11). In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs of the extracellular antigen-binding domain (e.g., in the FRs). In certain embodiments, the extracellular antigen-binding domain comprises a V-type amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 10, including post-translational modifications of these sequences (SEQ ID NO: 10 and SEQ ID NO: 11). H and V comprising the amino acid sequence shown in SEQ ID NO: 11 L Includes:

[0159] As used herein, the homology percentage between two amino acid sequences is equivalent to the identity percentage between two sequences.The identity percentage between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., homology%=number of identical positions / total number of positions×100).Comparing the sequences between two sequences and determining the identity percentage can be achieved using a mathematical algorithm.

[0160] The percent homology between two amino acid sequences was calculated using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, as incorporated into the ALIGN program (version 2.0) by E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) algorithm. Additionally, percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), as incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0161] Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can be further used as a "query sequence" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0162] 5.4.3.2. CAR Transmembrane Domain In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors cluster and a signal is transmitted to the cell. According to the subject matter of the present disclosure, the transmembrane domain of the CAR can comprise a natural or modified transmembrane domain of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.

[0163] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8).

[0164] In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence having NCBI reference number NP_001139345.1 (SEQ ID NO: 19) provided below (as used herein, homology can be determined using standard software such as BLAST or FASTA), and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 19 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO: 19. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or having amino acids 137-209 of SEQ ID NO: 19. [ka]

[0165] In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence having NCBI reference number AAA92533.1 (SEQ ID NO:20) provided below (as used herein, homology can be determined using standard software such as BLAST or FASTA), and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:20 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 20. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or having amino acids 151-219 of SEQ ID NO: 20. [ka]

[0166] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide that comprises or has the amino acid sequence set forth in SEQ ID NO: 21, provided below: STTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY [SEQ ID NO: 21]

[0167] According to the presently disclosed subject matter, a "CD8 nucleic acid molecule" refers to a polynucleotide that encodes a CD8 polypeptide.

[0168] In certain embodiments, an exemplary CD8 nucleic acid molecule encoding a CD8 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:21 is set forth in SEQ ID NO:22, provided below. [ka]

[0169] In certain embodiments, the transmembrane domain of a CAR of the present disclosure comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28).

[0170] A CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the sequence having NCBI reference number P10747 or NP_006130 (SEQ ID NO: 2), or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, a CD28 polypeptide comprises or consists of an amino acid sequence that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length of SEQ ID NO: 23. In various non-limiting embodiments, a CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 153-179, or 200-220 of SEQ ID NO: 23. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 23. SEQ ID NO: 23 is provided below: [ka]

[0171] According to the subject matter of this disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide that encodes a CD28 polypeptide.

[0172] In certain embodiments, an exemplary CD28 nucleic acid molecule encoding a CD28 polypeptide consisting of amino acids 153-179 of SEQ ID NO:23 is set forth in SEQ ID NO:24, provided below. ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg [SEQ ID NO: 24]

[0173] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25. SEQ ID NO: 25 is provided below: FWVLVVVGGV LACYSLLVTV AFIIFWV [SEQ ID NO: 25]

[0174] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:25 is shown in SEQ ID NO:26, provided below. TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG [SEQ ID NO: 26]

[0175] In certain non-limiting embodiments, the CAR further comprises a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be sufficiently flexible to allow the antigen-binding domain to orient in various directions to facilitate antigen recognition.

[0176] In certain non-limiting embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. The hinge / spacer region can be a hinge region from IgG1, or a portion of the CH2CH3 region and CD3 of an immunoglobulin, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 23), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 19 or a portion of SEQ ID NO: 20), a variant of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

[0177] 5.4.3.3. CAR Intracellular Signaling Domain In certain non-limiting embodiments, the CAR comprises an intracellular signaling domain. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("natural") CD3ζ contains three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2, ​​and BRS3). CD3ζ transmits activation signals to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the primary transmitter of signals from endogenous TCRs.

[0178] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence having NCBI reference number NP_932170 (SEQ ID NO: 27), or a fragment thereof, and / or optionally contains up to one, up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 27 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD3ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 27. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide comprising or having amino acids 52-164 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below: [ka]

[0179] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide is one disclosed in International Patent Publication No. WO2019 / 133969, which is hereby incorporated by reference in its entirety.

[0180] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:28 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. SEQ ID NO:28 is provided below: [ka]

[0181] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:28 is shown in SEQ ID NO:29, provided below. [ka]

[0182] In certain non-limiting embodiments, the intracellular signaling domain of CAR further comprises at least one costimulatory signaling region.In certain embodiments, the costimulatory region comprises at least one costimulatory molecule or a portion thereof.In certain embodiments, the costimulatory signaling region comprises the intracellular domain of at least one costimulatory molecule or a portion thereof.

[0183] As used herein, "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of lymphocytes to antigens. In certain embodiments, costimulatory molecules can provide optimal lymphocyte activation. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKGD2, CD2, and combinations thereof. A costimulatory molecule can bind to a costimulatory ligand, which is a protein expressed on the cell surface that generates a costimulatory response when binding to its receptor, i.e., an intracellular response that results in stimulation when an antigen-recognizing receptor (e.g., chimeric antigen receptor (CAR)) binds to its target antigen. As an example, 4-1BB ligand (i.e., 4-1BBL) in combination with a CAR signal stimulates the CAR + It can bind to 4-1BB to provide an intracellular signal that induces effector cell function in T cells.

[0184] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD28 polypeptide, e.g., a costimulatory signaling region comprising the intracellular domain of CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 23 or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 23 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 23. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 180-220 of SEQ ID NO: 23.

[0185] An exemplary nucleic acid sequence encoding amino acids 180-220 of SEQ ID NO:23 is shown in SEQ ID NO:30, provided below. [ka]

[0186] In certain embodiments, a CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the sequence having NCBI reference number NP_031668.3 (SEQ ID NO: 31), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, a CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-220, 150-200, 178-218, or 200-220 of SEQ ID NO: 31. In certain embodiments, the costimulatory signaling region of a CAR of the present disclosure comprises a CD28 polypeptide comprising or consisting of amino acids 178-218 of SEQ ID NO: 31. SEQ ID NO: 31 is provided below: [ka]

[0187] An exemplary nucleotide sequence encoding amino acids 178-218 of SEQ ID NO:31 is shown in SEQ ID NO:32, provided below. [ka]

[0188] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising two costimulatory molecules or portions thereof (e.g., the intracellular domains of costimulatory molecules), for example, the intracellular domains of CD28 and 4-1BB, or the intracellular domains of CD28 and OX40.

[0189] In certain non-limiting embodiments, the intracellular signaling domain of the CAR does not include a costimulatory signaling region, i.e., the CAR is a first-generation CAR. For example, the intracellular signaling domain of the CAR does not include the intracellular signaling domain of a costimulatory molecule, such as 4-1BB, CD28, etc. The costimulatory signaling domain contained in the CAR may cause uncontrolled proliferation. The activation of c-Kit contained in the cell can be controlled. For example, as disclosed in Section 5.2, the c-Kit mutant may be operably linked to a promoter, such as an inducible promoter, that can control the activation of c-Kit, and for example, by controlling the inducible promoter, c-Kit is activated only upon activation of cells (e.g., T cells or CAR-T cells) containing the c-Kit mutant.

[0190] 5.4.3.4. Example CAR In certain embodiments, the CAR is a mesothelin-targeting CAR. In certain embodiments, the CAR comprises (a) a V having the amino acid sequence set forth in SEQ ID NO: 4. H CDR1, V having the amino acid sequence shown in SEQ ID NO:5 H CDR2, V having the amino acid sequence shown in SEQ ID NO:6 H CDR3, V having the amino acid sequence shown in SEQ ID NO:7 L CDR1, V having the amino acid sequence shown in SEQ ID NO:8 L CDR2, and V having the amino acid sequence shown in SEQ ID NO:9 L (b) an extracellular antigen-binding domain comprising a CDR3; (b) a transmembrane domain comprising the transmembrane domain of CD28; and (c) an intracellular signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of CD28).

[0191] In certain embodiments, the CAR is a mesothelin-targeting CAR. In certain embodiments, the CAR comprises (a) a VH CDR1, V consisting of the amino acid sequence shown in SEQ ID NO: 5 H CDR2, V consisting of the amino acid sequence shown in SEQ ID NO:6 H CDR3, V consisting of the amino acid sequence shown in SEQ ID NO:7 L CDR1, V consisting of the amino acid sequence shown in SEQ ID NO: 8 L CDR2, and V consisting of the amino acid sequence shown in SEQ ID NO: 9 L (b) an extracellular antigen-binding domain comprising a CDR3; (b) a transmembrane domain comprising the transmembrane domain of CD8; and (c) an intracellular signaling domain comprising a CD3ζ polypeptide, but not a costimulatory signaling region.

[0192] In certain embodiments, the CAR of the present disclosure further comprises an inducible promoter for expressing the nucleic acid sequence in human cells. The promoter for use in expressing the CAR gene can be a constitutive promoter, such as the ubiquitin C (UbiC) promoter.

[0193] 5.4.4. TCR-like fusion molecules In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also known as "HIT-CARs," e.g., those disclosed in International Patent Application No. PCT / US19 / 017525, the entirety of which is incorporated herein by reference) and T cell receptor fusion constructs (TRuCs) (e.g., those disclosed in Baeuerle et al., "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response," Nature Communications volume 10, Article number: 2087, the entirety of which is incorporated herein by reference). (2019)).

[0194] The presently disclosed subject matter provides polypeptide compositions comprising a mesothelin-targeting chimeric antigen receptor (CAR) and a dominant-negative form of programmed death 1 (PD-1 DN).

[0195] 5.5. Dominant Negative Programmed Death 1 (PD-1 DN) In certain embodiments, the cells of the presently disclosed subject matter further comprise a dominant-negative form of programmed death 1 (referred to as "PD-1 DN").

[0196] PD-1 DN can enhance the therapeutic efficacy of immunoresponsive cells, including CARs. In certain embodiments, the PD-1 DN comprises (a) at least a portion of the extracellular domain of programmed death 1 (PD-1), including the ligand-binding region, and (b) the transmembrane domain.

[0197] In certain embodiments, cells, e.g., T cells, are engineered to express a dominant-negative form (DN form) of PD-1.

[0198] Malignant cells adapt to generate an immunosuppressive microenvironment that protects them from immune recognition and elimination (Sharpe et al., Dis. Model Mech. 8:337-350 (2015)). The immunosuppressive microenvironment imposes limitations on immunotherapy methods. Details of inhibitors of DN forms of cell-mediated immune responses are disclosed in WO2017 / 040945 and WO2017 / 100428, the contents of each of which are incorporated herein by reference in their entirety.

[0199] Programmed cell death protein 1 (PD-1) is a negative immune regulator of activated T cells through binding to its corresponding ligands, PD-L1 and PD-L2, which are expressed on endogenous macrophages and dendritic cells. PD-1 is a 268-amino acid type I membrane protein. PD-1 consists of two ligands, PD-L1 and PD-L2, which are members of the B7 family. The protein structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located within an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif. PD-1 negatively regulates TCR signaling. SHP-1 and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism used by tumor cells to evade the host immune system. In preclinical and clinical studies, PD-1 blockade with antagonistic antibodies induced antitumor responses mediated through the host's endogenous immune system.

[0200] In certain embodiments, the PD-1 polypeptide consists of the amino acid sequence having GenBank No. NP_005009.2 (SEQ ID NO: 33) or a fragment thereof. In certain embodiments, amino acids 1-20 of SEQ ID NO: 33 are the signal peptide (or peptide signal) of PD-1. In certain embodiments, amino acids 21-170 of SEQ ID NO: 33 are the extracellular domain of PD-1. In certain embodiments, amino acids 171-191 of SEQ ID NO: 33 are the transmembrane domain of PD-1. In certain embodiments, amino acids 192-288 of SEQ ID NO: 33 are the intracellular domain of PD-1. SEQ ID NO: 33 is provided below: [ka]

[0201] In certain embodiments, the extracellular domain of PD-1 comprises a ligand-binding domain (referred to as the "extracellular ligand-binding domain"). In certain embodiments, the extracellular ligand-binding domain of PD-1 is fused to one or more heterologous polypeptide sequences, i.e., the PD-1 DN is a chimeric sequence. For example, the extracellular ligand-binding domain of PD-1 can be fused at its N-terminus to a signal peptide, which is optionally a heterologous signal peptide, including various signal peptides described herein. Furthermore, the PD-1 DN can comprise a transmembrane domain, which is optionally a heterologous transmembrane domain, including any of the various transmembrane domains described herein.

[0202] In certain embodiments, PD-1 DN comprises the extracellular domain of a PD-1 polypeptide (e.g., amino acids 21-170 of SEQ ID NO: 33) or a ligand-binding portion thereof (e.g., amino acids 21-165 of SEQ ID NO: 33). Cells expressing such PD-1 DN may lack or have reduced ability to signal in the PD-1 immune checkpoint pathway. In certain embodiments, PD-1 DN is a deletion mutant (e.g., PD-1 DN lacking amino acids 192-288 of SEQ ID NO: 33) or a portion thereof, lacking the intracellular domain. PD-1 with a deletion of the intracellular domain may have reduced or inhibited PD-1-mediated immune checkpoint pathways.

[0203] In certain embodiments, the PD-1 DN comprises the extracellular ligand-binding domain of PD-1. In certain embodiments, the PD-1 DN comprises the extracellular ligand-binding domain of a PD-1 polypeptide and the transmembrane domain of a PD-1 polypeptide. In certain embodiments, the PD-1 DN comprises or consists of amino acids 21-165 of SEQ ID NO: 33.

[0204] An exemplary nucleotide sequence encoding amino acids 21-165 of SEQ ID NO:33 is shown in SEQ ID NO:34, provided below. [ka] [ka]

[0205] In certain embodiments, the PD-1 DN further comprises a signal peptide, e.g., the PD-1 DN comprises the extracellular ligand-binding domain of a PD-1 polypeptide, the transmembrane domain of a PD-1 polypeptide, and the signal peptide of a PD-1 polypeptide. In certain embodiments, the signal peptide comprises or consists of amino acids 1-20 of SEQ ID NO: 33. An exemplary nucleotide sequence encoding amino acids 1-20 of SEQ ID NO: 33 is set forth in SEQ ID NO: 35, provided below. ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGG [SEQ ID NO: 35]

[0206] In certain embodiments, the PD-1 DN comprises or consists of amino acids 1-165 of SEQ ID NO:33.

[0207] An exemplary nucleotide sequence encoding amino acids 1-165 of SEQ ID NO:33 is shown in SEQ ID NO:36, provided below. [ka]

[0208] In certain embodiments, the PD-1 DN comprises or consists of amino acids 21-151 of SEQ ID NO: 33. In certain embodiments, the PD-1 DN comprises or consists of amino acids 1-151 of SEQ ID NO: 33. In certain embodiments, the PD-1 The DN comprises or consists of amino acids 21-151 of SEQ ID NO: 33. In certain embodiments, the PD-1 DN comprises or consists of the amino acid sequence beginning at amino acid 21 of SEQ ID NO: 33 and extending to between amino acids 151-165 of SEQ ID NO: 33.

[0209] In certain embodiments, the PD-1 DN further comprises a CD8 polypeptide. In certain embodiments, the PD-1 DN comprises the extracellular domain of PD-1 or a portion thereof (e.g., the extracellular ligand-binding domain) fused to the transmembrane domain and / or hinge domain of CD8. In certain embodiments, the PD-1 DN comprises the transmembrane domain of CD8 (e.g., amino acids 183-203 of SEQ ID NO: 19). Such embodiments represent chimeric DN forms comprising transmembrane domains derived from different (heterologous) polypeptides. As described above, PD-1 DNs comprising heterologous domains, e.g., transmembrane domains, can optionally comprise additional sequences derived from the heterologous polypeptide. In certain embodiments, the PD-1 DN comprises additional sequences N-terminal to the transmembrane domain from the heterologous polypeptide. In certain embodiments, the PD-1 DN comprises the hinge domain of CD8. In certain embodiments, the heterologous sequence comprises additional N-terminal sequence of a CD8 polypeptide (e.g., amino acids 137-182 (or optionally starting at amino acids 138 or 139) of SEQ ID NO: 19). In certain embodiments, the PD-1 DN comprises additional sequence from a heterologous polypeptide C-terminal to the CD8 transmembrane domain. In certain embodiments, the additional C-terminal sequence is amino acids 204-209 of SEQ ID NO: 19.

[0210] In certain embodiments, the PD-1 DN comprises the transmembrane domain of a CD8 polypeptide (e.g., amino acids 183-203 of SEQ ID NO: 19), the hinge domain of a CD8 polypeptide (e.g., amino acids 137-182 of SEQ ID NO: 19), and additional C-terminal sequence of a CD8 polypeptide (e.g., amino acids 204-207 of SEQ ID NO: 19). In certain embodiments, the PD-1 DN comprises a CD8 polypeptide consisting of amino acids 137-207 of SEQ ID NO: 19.

[0211] An exemplary nucleotide sequence encoding amino acids 137-207 of SEQ ID NO:19 is shown in SEQ ID NO:37, provided below: [ka]

[0212] In certain embodiments, the PD-1 DN comprises the transmembrane domain of a CD8 polypeptide (e.g., amino acids 183-203 of SEQ ID NO: 19), the hinge domain of a CD8 polypeptide (e.g., amino acids 137-182 of SEQ ID NO: 19), and additional C-terminal sequence of a CD8 polypeptide (e.g., amino acids 204-209 of SEQ ID NO: 19). In certain embodiments, the PD-1 DN comprises a CD8 polypeptide having amino acids 137-209 of SEQ ID NO: 19.

[0213] An exemplary nucleotide sequence encoding amino acids 137-209 of SEQ ID NO:19 is shown in SEQ ID NO:38, provided below: [ka]

[0214] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO: 39, provided below. [ka] [ka]

[0215] An exemplary nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:39 is set forth in SEQ ID NO:40, provided below: [ka]

[0216] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO:41, provided below. [ka]

[0217] An exemplary nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:41 is set forth in SEQ ID NO:42, provided below: [ka]

[0218] In certain non-limiting embodiments, the transmembrane domain of PD-1 DN comprises a hydrophobic alpha helix spanning at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. According to the presently disclosed subject matter, the transmembrane domain of PD-1 DN can comprise the native or modified transmembrane domain of any polypeptide disclosed herein, for example, any transmembrane domain that can be contained in a chimeric antigen receptor. In certain embodiments, the transmembrane domain is a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 peptide, an NKGD2 peptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. In certain embodiments, the transmembrane domain is a CD8 polypeptide. Details of these transmembrane domains are described in Section 5.4.

[0219] 5.6. Compositions and Vectors The presently disclosed subject matter provides (a) a composition comprising a c-Kit mutant (e.g., as disclosed in Section 5.2) disclosed herein and an antigen-recognizing receptor (e.g., as disclosed in Section 5.4) disclosed herein. Cells comprising such compositions are also provided.

[0220] In certain embodiments, the c-Kit mutant is operably linked to a first promoter, hi certain embodiments, the antigen-recognizing receptor is operably linked to a second promoter.

[0221] Furthermore, the presently disclosed subject matter provides a nucleic acid composition comprising a first polynucleotide encoding a c-Kit variant disclosed herein (e.g., as disclosed in Section 5.2) and a second polynucleotide encoding an antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 5.4). Cells comprising such nucleic acid compositions are also provided.

[0222] In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the c-Kit mutant, hi certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to an antigen-recognizing receptor.

[0223] In certain embodiments, one or both of the first and second promoters are endogenous or exogenous.In certain embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF)-1 promoter, CMV promoter, SV40 promoter, PGK promoter, and metallothionein promoter.In certain embodiments, one or both of the first and second promoters are inducible promoters.In certain embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0224] The compositions and nucleic acid compositions can be administered to a subject, or can be delivered into cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transducing a recombinant DNA construct into a substantially homogeneous cell composition. In certain embodiments, retroviral vectors (e.g., gamma retroviral vectors or lentiviral vectors) are used to introduce DNA constructs into cells. For example, a polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific to the target cell type of interest. Non-viral vectors can also be used.

[0225] For the initial genetic modification of cells to contain an antigen-recognizing receptor (e.g., CAR or TCR), retroviral vectors are generally used for transduction, but any other suitable viral vector or non-viral delivery system can also be used. The antigen-recognizing receptor and c-Kit mutant can be constructed in a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Plague virus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). The combination of a retroviral vector with an appropriate packaging system is also suitable when the capsid protein is functional for infecting human cells. A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. (1985); 5:431-437); PA317 (Miller, et al. Mol. Cell. Biol. (1986); 6:2895-2902); and CRIP (Danos, et al. Proc. Natl. Acad. Sci. USA (1988); 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114, or GALV envelopes and any others known in the art, are also suitable.

[0226] Possible methods of transduction include direct co-culture of cells with producer cells (Bregni, et al. Blood (1992); 80:1418-1422) or with viral supernatant alone or in the presence of appropriate growth factors and polycations (Xu, et al. Exp. Hemat. (1994); 22:223-230; and Hughes, et al. J. Clin. Invest. (1992); 89:1817) with or without concentrated vector stocks.

[0227] Other transducing viral vectors may be used to modify immunoresponsive cells. In certain embodiments, the vector selected exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated virus vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., J. Immunol. 1999, 114:1277-1278, 1991). al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987;Anderson, Science 226:401-409, 1984;Moen, Blood Cells 17:407-416, 1991;Miller et al., Biotechnology 7:980-990, 1989;LeGal La Salle et al. (See also the vectors in Rosenberg et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995.) Retroviral vectors are particularly well developed and are used in clinical practice (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0228] Non-viral approaches can also be used to genetically modify immunoresponsive cells, for example, by administering nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters Nucleic acid molecules can be introduced into immunocompetent cells by asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for the delivery of DNA into cells.The transplantation of normal genes into the affected tissues of a subject can be achieved by transferring normal nucleic acids into ex vivo cultivable cell types (for example, autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into target tissues or systemically injecting them.Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (for example, zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR).Transient expression can be obtained by RNA electroporation.

[0229] Any targeted genome editing method can be used to deliver the c-Kit mutant and / or antigen-recognizing receptor disclosed herein to cell or subject.In certain embodiments, CRISPR system is used to deliver the c-Kit mutant and / or antigen-recognizing receptor disclosed herein.In certain embodiments, zinc finger nuclease is used to deliver the c-Kit mutant and / or antigen-recognizing receptor disclosed herein.In certain embodiments, TALEN system is used to deliver the c-Kit mutant and / or antigen-recognizing receptor disclosed herein.

[0230] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, this system contains Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA, along with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process, allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 is often transfected using a plasmid into target cells. The crRNA is the sequence used by Cas9 to identify and directly bind to the target DNA in the cell, so it needs to be designed for each application. The repair template carrying the CAR expression cassette also needs to be designed for each application, as it must overlap with the sequences on either side of the cut and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA), which can be spliced ​​with a Cas9 gene, made into a plasmid, and transfected into cells.

[0231] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger nuclease to target desired sequences within the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is to combine zinc finger "modules" of known smaller specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) mechanism and a homologous DNA template carrying a CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. Once the targeting sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template and then copies the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0232] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems operate on much the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain. Transcription activator-like effectors (TALEs) consist of a 33-34 amino acid repeat motif with two variable positions that have strong recognition for specific nucleotides. By assembling an array of these TALEs, the TALE DNA-binding domain can be engineered to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location within the genome. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and regulated by any appropriate mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of nucleic acid.The enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers.Alternatively, when genomic clones are used as therapeutic constructs, regulation can be mediated by homologous regulatory sequences, including any of the promoters or regulatory elements described above, or, if desired, by regulatory sequences from heterologous sources.

[0233] The method for delivering genome editing agents / systems can vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered by viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, pseudotyped viral vector envelope proteins, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0234] 5.7. Polypeptides and Analogs The presently disclosed subject matter also includes mesothelin, CD28, CD8, CD3ζ, and c-Kit polypeptides or fragments thereof that have been modified in a manner that enhances their antineoplastic activity when expressed in immunoresponsive cells. The presently disclosed subject matter provides methods for optimizing amino acid or nucleic acid sequences by making changes in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter also includes analogs of any naturally occurring polypeptide disclosed herein. Analogs may differ from the naturally occurring polypeptides disclosed herein (including, but not limited to, mesothelin, CD28, CD8, CD3ζ, and c-Kit) by differences in amino acid sequence, by post-translational modifications, or both. Analogs may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology to all or a portion of the naturally occurring amino acid sequence of the subject matter of the present disclosure. The length of sequence comparison is at least 5, 10, 15, or 20 amino acid residues, e.g., at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Again, an exemplary approach to determining the degree of identity can use the BLAST program, e.g., -3 and e -100 A probability score between indicates closely related sequences. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogs may also differ from naturally occurring polypeptides by changes in primary sequence. These include both natural and induced genetic variants (e.g., resulting from random mutagenesis by irradiation or exposure to ethane methyl sulfate, or by the methods of Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al. al., supra.) Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as, for example, D-amino acids or non-naturally occurring or synthetic amino acids (e.g., β or γ amino acids).

[0235] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptide or peptide domains disclosed herein. As used herein, the term "fragment" refers to at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60-80, 100, 200, 300, or more contiguous amino acids. Fragments may be generated by methods known to those of skill in the art or may result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0236] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins (e.g., c-Kit mutants) disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide. Methods for analog design are well known in the art, and analog synthesis can be performed according to such methods by modifying the chemical structure to increase the antineoplastic activity of the original polypeptide when the resulting analog is expressed in immunoresponsive cells. These chemical modifications include, but are not limited to, substituting alternative R groups and changing the degree of saturation at specific carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to in vivo degradation and provide a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0237] Administration The presently disclosed subject matter also provides compositions comprising the cells of the present disclosure.

[0238] Compositions comprising the cells of the present disclosure may be administered systemically or directly to a subject to induce and / or enhance an immune response to an antigen and / or treat and / or prevent a neoplasm, pathogen infection, or infectious disease. In certain embodiments, the cells of the present disclosure or a composition comprising them are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the cells of the present disclosure or a composition comprising them are administered indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the vasculature of a tumor). Expansion and differentiation agents can be administered before, during, or after administration of the cells or composition to increase the generation of T cells or NK cells in vitro or in vivo.

[0239] The cells of the present disclosure can be administered in any physiologically acceptable vehicle, usually intravascularly, but they can also be introduced into bone or other convenient sites (e.g., the thymus) where the cells can find a suitable site for regeneration and differentiation. Typically, at least about 1 x 10 5 cells were administered, resulting in a final total of approximately 1 × 10 10 The cells of the present disclosure may comprise a purified population of cells. Those skilled in the art can easily determine the percentage of cells of the present disclosure in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing immunoresponsive cells of the present disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. Dosage can be easily adjusted by those skilled in the art (e.g., a decrease in purity may require an increased dosage). Cells can be introduced by injection, catheter, etc.

[0240] The composition of the present disclosure may be a pharmaceutical composition comprising the cells of the present disclosure and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, cells may be obtained from one subject and administered to the same subject or a different, compatible subject. Peripheral blood-derived cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) may be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering a composition of the present disclosure (e.g., a pharmaceutical composition comprising the cells of the present disclosure), it may be formulated into an injectable unit dosage form (solution, suspension, emulsion).

[0241] In certain embodiments, the composition further comprises an inhibitor of c-Kit (referred to as a "c-Kit inhibitor"). The c-Kit inhibitor can inhibit the activity (e.g., kinase activity) of c-Kit. In certain embodiments, the c-Kit specifically inhibits a c-Kit mutant, for example, c-Kit D816V. In certain embodiments, the c-Kit inhibitor is a multi-tyrosine kinase inhibitor. Non-limiting examples of c-Kit inhibitors include dasatinib, midostaurin, ponatinib, and imatinib.

[0242] 5.9. Formulations Compositions containing the cells of the present disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions (which may be buffered to a selected pH). Liquid preparations are typically easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to achieve longer contact periods with specific tissues. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0243] Sterile injectable solutions can be prepared by incorporating the genetically modified cells in the required amount of an appropriate solvent, along with various amounts of other ingredients, if desired. Such compositions may be mixed with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, etc. The compositions may also be lyophilized. The compositions may contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the desired route of administration and preparation. Reference may be made to standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985, incorporated herein by reference, to prepare suitable preparations without undue experimentation.

[0244] Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with genetically modified cells.

[0245] The compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as glucose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be used, particularly for buffers containing sodium ions.

[0246] If desired, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. For example, methylcellulose is readily and economically available and easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening agent can depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. Obviously, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of the specific dosage form, for example, a liquid dosage form (for example, whether the composition should be formulated into a solution, suspension, gel, or another liquid form, such as a time-release form or liquid-filled form).

[0247] The amount of cells administered will vary depending on the subject being treated. In one embodiment, approximately 10 of the immunoresponsive cells of the present disclosure are administered. 4 ~about 10 10 Between pieces, approximately 10 4 ~about 10 6 Between pieces, approximately 10 5 ~about 10 9 Between pieces, or about 10 6 ~about 10 8 Between about 1 x 10 cells are administered to a human subject. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1 x 10 cells of the present disclosure are administered. 5 , about 2×10 5 , about 3×10 5 , about 4×10 5 , or about 5 × 10 5 The dosage is administered to a human subject.The exact determination of what is considered to be an effective dose may be based on factors specific to each subject, including their size, age, sex, weight, and the condition of a particular subject.Dosage can be easily ascertained by those skilled in the art from this disclosure and knowledge in the art.

[0248] Those skilled in the art can readily determine the amounts of cells and optional additives, vehicles, and / or carriers to be administered in the compositions and methods. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of 0.001 to 50% (by weight) solution in phosphate-buffered saline, with the active ingredient being present on the order of micrograms to milligrams, e.g., about 0.0001 to about 5 wt%, about 0.0001 to about 1 wt%, about 0.0001 to about 0.05 wt%, or about 0.001 to about 20 wt%, about 0.01 to about 10 wt%, or about 0.05 to about 5 wt%. For any composition to be administered to animals or humans, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., a rodent such as a mouse; the dosage of the composition(s) that induces a suitable response, the concentration of the components therein, and the timing of administering the composition(s). Such determinations can be made without undue experimentation by those skilled in the art, based on the knowledge of the present disclosure and the documents cited herein. And the time for sequential administration can be ascertained without undue experimentation.

[0249] 5.10. Treatment Method The subject cells of the present disclosure and compositions comprising same can be used for the treatment and / or prevention of neoplasms, pathogen infections, infectious diseases, inflammatory diseases, or transplant rejection. Such cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment or prevention of solid tumors (e.g., mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, and / or bile duct cancer). In certain embodiments, the cells are T cells. T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + T cells.

[0250] In certain embodiments, neoplastic or tumor cells have high expression levels of mesothelin (referred to as "high MSLN-expressing cells"). In certain embodiments, high MSLN-expressing cells are cells that express mesothelin at an expression level of about 50 times or more, about 50 times or more, about 60 times or more, about 70 times or more, about 80 times or more, about 90 times or more, about 100 times or more, about 150 times or more, about 200 times or more, about 300 times or more, about 400 times or more, or about 500 times or more compared to the mesothelin expression level of normal cells.

[0251] In certain embodiments, cells of a neoplasm or tumor have low expression levels of mesothelin (referred to as "low-MSLN-expressing cells"). In certain embodiments, low-MSLN-expressing cells are cells that express mesothelin at an expression level that is about 50-fold or less, about 40-fold or less, about 30-fold or less, about 20-fold or less, about 10-fold or less, about 5-fold or less, about 4-fold or less, about 3-fold or less, or about 2-fold or less compared to the mesothelin expression level of normal cells.

[0252] In certain embodiments, the solid tumor is lung cancer.

[0253] In certain embodiments, the solid tumor is mesothelioma.In certain embodiments, the mesothelioma cell is a high MSLN expression cell.In certain embodiments, the cell used in the treatment of high MSLN expression mesothelioma cell comprises a CAR (for example, a first-generation CAR) that does not include a costimulatory signaling region.

[0254] The presently disclosed subject matter provides methods for inducing and / or increasing an immune response in a subject in need thereof. The cells of the present disclosure and compositions comprising the same can be used in therapy or medicine. The cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent neoplasms in a subject. The cells of the present disclosure and compositions comprising the same can be used to prolong the survival time of a subject suffering from a neoplasm. The cells of the present disclosure and compositions comprising the same can also be used to treat and / or prevent pathogen infection or other infectious diseases in a subject, for example, an immunocompromised human subject. Such methods include administering the cells or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect (even if it is alleviation of an existing condition or prevention of recurrence). For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be administered in one or a series of administrations. An effective amount can be administered as a bolus or by continuous perfusion.

[0255] An "effective amount" (i.e., a "therapeutically effective amount") is an amount sufficient to produce beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disease, or otherwise reduce the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of a person skilled in the art. When determining an appropriate dosage to achieve an effective amount, several factors are typically taken into consideration. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells to be administered.

[0256] Adoptive immunotherapy, which uses antigen-specific T cells, produces approximately 10 6 ~10 10 (e.g., about 10 9) cell doses are typically injected. Upon administration of the cells of the present disclosure to a host and subsequent differentiation, T cells specifically directed against a specific antigen are induced. The modified cells may be administered by any method known in the art, including, but not limited to, intrapleural administration, intravenous administration, subcutaneous administration, intralymph node administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration into the thymus. In certain embodiments, the immunoresponsive cells and compositions comprising the same are administered intrapleurally to a subject in need thereof. The presently disclosed subject matter provides various methods of using cells (e.g., T cells) or compositions comprising the same. For example, the presently disclosed subject matter provides a method of reducing tumor burden in a subject. In certain embodiments, the method of reducing tumor burden comprises administering the cells of the present disclosure or a composition comprising the same to a subject. The cells of the present disclosure can reduce the number of tumor cells, decrease tumor size, and / or eradicate tumors in a subject. The tumor may be a solid tumor. Non-limiting examples of solid tumors include mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, and bile duct carcinoma.

[0257] The presently disclosed subject matter also provides a method for increasing or extending the survival time of a subject having a neoplasm. In certain embodiments, the method for increasing or extending the survival time of a subject having a neoplasm comprises administering to the subject an effective amount of an immunoresponsive cell of the present disclosure or a composition comprising the same. The method can reduce or eradicate tumor burden in the subject. Furthermore, the presently disclosed subject matter provides a method for increasing an immune response in a subject, the method comprising administering to the subject a cell of the present disclosure or a composition comprising the same. The presently disclosed subject matter further provides a method for treating and / or preventing a neoplasm in a subject, the method comprising administering to the subject a cell of the present disclosure or a composition comprising the same.

[0258] As used herein, the term "neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration to or invasion of other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell proliferation and proliferation. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, organs selected from the group consisting of the bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or tissues or cell types thereof. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In one embodiment, the neoplasm is a solid tumor. The neoplasm can be a primary tumor or primary cancer. Additionally, the neoplasm may be in a metastatic state.

[0259] Cancers whose growth can be inhibited using the immunoresponsive cells of the presently disclosed subject matter include cancers that are typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include mesothelioma, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (e.g., metastatic breast cancer, metastatic triple-negative breast cancer), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, bile duct cancer, cervical cancer, and salivary gland cancer. Additionally, the presently disclosed subject matter includes resistant or recurrent malignant tumors whose growth can be inhibited using the immunoresponsive cells of the presently disclosed subject matter.

[0260] Examples of other neoplasms or cancers that can be treated using the methods of the presently disclosed subject matter include bone cancer, intestinal cancer, liver cancer, skin cancer, head or neck cancer, melanoma (cutaneous or intraocular malignant melanoma), kidney cancer (e.g., clear cell carcinoma), pharyngeal cancer, prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), blood cancer (e.g., leukemia, lymphoma, and myeloma), uterine cancer, rectal cancer, cancer of the anal region, bladder cancer, brain cancer, stomach cancer, testicular cancer, fallopian tube cancer, and the like. Cancer, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland , soft tissue sarcomas, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, Waldenstrom's macroglobulinemia, heavy chain disease, etc. and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, cholangiocarcinoma, duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0261] The subject may have an advanced form of the disease, in which case the goal of treatment may include reducing or reversing disease progression and / or ameliorating side effects. The subject may have a history of a condition that has already been treated, in which case the goal of treatment typically includes reducing or delaying the risk of recurrence.

[0262] Human subjects suitable for treatment typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are subjects with clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; positive biochemical or histopathological markers themselves are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to induce an anti-tumor response with the aim of alleviating these conditions. Ideally, a reduction in tumor mass occurs as a result, but any clinical improvement also constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of progression, or a reduction in the pathological consequences of tumors.

[0263] Suitable subjects in the second group are known in the art as the "adjuvant group." These are individuals who have a history of neoplasia but have responded to other treatment modalities. Previous treatments may include, but are not limited to, surgical resection, radiation therapy, and traditional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected of being at risk for disease progression, either near the original tumor site or through metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is based on features observed before and after initial treatment. These features are known in the clinical arts and are appropriately defined for different neoplasms. Typical features of the high-risk subgroup are tumor invasion into adjacent tissues or lymph node involvement.

[0264] Another group has a genetic predisposition to neoplasia but has not yet manifested clinical signs of the neoplasia. For example, a woman who tests positive for a genetic mutation associated with breast cancer but is still of childbearing age may wish to receive one or more of the immunoresponsive cells described in the present invention in a prophylactic treatment to prevent the appearance of a neoplasia until it is appropriate to perform prophylactic surgery.

[0265] As a result of the expression of antigen-recognition receptors that bind to tumor antigens and c-Kit mutants that enhance the antitumor effects of immunocompetent cells, adoptively transferred T or NK cells are endowed with increased selective cytolytic activity at the tumor site. Furthermore, following their localization to the tumor or viral infection and their proliferation, T cells transform the tumor or viral infection site into a highly conducive environment for a wide range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) that participate in physiological antitumor or antiviral responses.

[0266] Furthermore, the presently disclosed subject matter provides a method for treating and / or preventing a pathogen infection (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoan infection) in a subject, for example, an immunocompromised subject. The method may include administering an effective amount of a cell of the present disclosure or a composition comprising the same to a subject with a pathogen infection. Exemplary viral infections amenable to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infection.

[0267] Further modifications can be introduced into the immunoresponsive cells (e.g., T cells) of the present disclosure to avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or the risk of an outcome similar to GvHD if healthy tissue expresses the same target antigen as tumor cells. A potential solution to this problem is to engineer a suicide gene into the immunoresponsive cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase-9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can enable T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt may be covalently conjugated upstream of the antigen-recognition receptor of the CAR of the present disclosure. The suicide gene may be included in a vector containing a nucleic acid encoding a CAR of the present disclosure. In this method, administration of a prodrug (e.g., a prodrug (e.g., AP1903, which can activate iCasp-9)) designed to activate the suicide gene during malignant T cell transformation (e.g., GVHD) induces apoptosis in suicide gene-activated CAR-expressing T cells. Incorporation of a suicide gene into a CAR of the present disclosure confers an additional level of safety with the ability to eliminate the majority of CAR T cells within a very short period of time. Immunoreactive cells (e.g., T cells) of the present disclosure that have incorporated a suicide gene can be preemptively eliminated at a given time point after CAR T cell infusion or eradicated at the earliest sign of toxicity.

[0268] Furthermore, the presently disclosed subject matter provides a method for preventing and / or treating an inflammatory disease in a subject. In certain embodiments, the method comprises administering to a subject a cell of the present disclosure or a composition comprising the same. In certain embodiments, the cell is an immunoinhibitory cell. In certain embodiments, the immunoinhibitory cell is a regulatory T cell. In one embodiment, the inflammatory disease is pancreatitis. In certain embodiments, the subject is a human. In certain embodiments, the subject is an organ transplant recipient, for example, a pancreas transplant recipient.

[0269] Furthermore, the subject matter of the present disclosure provides a method for preventing graft rejection in a subject who is a recipient of an organ transplant. In certain embodiments, the method comprises administering to the subject a cell of the present disclosure or a composition comprising the same. In certain embodiments, the cell is an immunosuppressive cell. In certain embodiments, the immunosuppressive cell is a regulatory T cell. In certain embodiments, the subject is a human. In further embodiments, the subject is a recipient of a pancreatic transplant.

[0270] In certain embodiments, the methods disclosed herein further include administering to the subject a c-Kit inhibitor (e.g., one disclosed in Section 5.8). The c-Kit inhibitor can inhibit the activity (e.g., kinase activity) of c-Kit. In certain embodiments, the c-Kit inhibitor specifically inhibits a c-Kit mutant, for example, c-Kit D816V. In certain embodiments, the c-Kit inhibitor is a multi-tyrosine kinase inhibitor. Non-limiting examples of c-Kit inhibitors include dasatinib, midostaurin, ponatinib, and imatinib. 5.11.Kit

[0271] The subject matter of the present disclosure provides a kit for inducing and / or enhancing an immune response in a subject and / or treating and / or preventing a neoplasm, pathogen infection, or immune disorder. In certain embodiments, the kit includes a cell of the present disclosure or a composition comprising the cell. In certain embodiments, the kit includes a sterile container; such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit includes an isolated nucleic acid molecule encoding an antigen-recognition receptor (e.g., CAR or TCR) for an antigen of interest and an isolated nucleic acid molecule encoding a c-Kit mutant in an expressible form (which may be contained in the same or different vectors, as needed).

[0272] If desired, the cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing a neoplasm or pathogen immune disorder. The instructions generally include information about using the composition to treat or prevent a neoplasm or pathogen infection. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; dosing schedule and administration for treating or preventing a neoplasm, pathogen infection, or immune disorder or its symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or may be printed as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. [Example]

[0273] 6. Working Example The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and therefore may be considered in making and practicing the presently disclosed subject matter. Particularly useful techniques for certain embodiments are discussed in the following sections.

[0274] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed cells and compositions, and are not intended to limit the scope of what the inventors regard as their invention.

[0275] Example 1 Generation of constructs Two constructs of the presently disclosed subject matter were generated. One construct is designated "M28z-KITv" (also referred to as "M28z-KITm"). The structure of M28z-KITv is shown in FIG. 1A. As shown in FIG. 1A, M28z-KITv comprises a c-Kit mutant (e.g., c-Kit D816V) and a second-generation CAR comprising an anti-MSKN scFv, a transmembrane domain comprising a CD28 polypeptide (e.g., a CD28 transmembrane domain or a portion thereof), and an intracellular domain comprising CD3ζ and a CD28 polypeptide (e.g., the intracellular domain of CD28). Another construct is designated "Mz-KITv." The structure of Mz-KITv is shown in FIG. 1B. As shown in Figure 1B, Mz-KITv comprises a c-Kit mutant (e.g., c-Kit D816V) and a first-generation CAR comprising an anti-MSKN scFv, a transmembrane domain comprising a CD28 polypeptide (e.g., the CD28 transmembrane domain or a portion thereof), and an intracellular domain comprising CD3ζ.

[0276] Example 2 Transduction of constructs, activation of pKIT signaling, expansion and proliferation The transduction, activation of pKIT signaling, expansion, and proliferation of M28z-KITv and Mz-KITv were examined. M28z contains the same CAR construct as M28z-KITv, but does not contain the Kit mutant. The transduction results are shown in Figure 2. As shown in Figure 2, both M28z-KITv and Mz-KITv had good transduction. The MFI value for T cells containing M28z was higher than that of T cells containing M28z-KITv (referred to as "M28z-KITv CAR T cells") or T cells containing Mz-KITv (referred to as "Mz-KITv CAR T cells").

[0277] The results of the constructs for their ability to activate pKIT signaling are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, M28z-KITv CAR T cells constitutively exhibited activated pKIT signaling. As shown in Figure 3A, M28z-KITv CAR T cells exhibited pKIT activity without SCF, whereas M28z CAR T cells did not express KIT protein. As shown in Figure 3B, M28z-KITv CAR T cells exhibited higher p-STAT3 and p-STAT5 activity than the M28z-KITwt CAR T cell control.

[0278] The CAR T cell expansion results are shown in Figure 4. Figure 4 shows the cumulative expansion of CAR T cells during continuous coculture. T cells were restimulated with A549GM tumor cells (E:T = 3:1). As shown in Figure 4, M28z-KITv CAR T cells and M28z CAR T cells showed similar expansion levels, while Mz-KITv CAR T cells showed less expansion. A549GM was a non-small cell lung cancer cell line with overexpression of GFP-luciferase and mesothelin.

[0279] The proliferation results are shown in Figures 5 and 6. Far red cell trace staining of CAR T cells is measured 7 days after the first antigen stimulation (E:T=2:1). The target cells were A549GM. As shown in Figures 5 and 6, M28z-KITm CAR The T cells showed higher proliferation compared to M28z CAR T cells.

[0280] Thus, the cKIT costimulatory construct demonstrated successful transduction and antigen-specific activation and proliferation.

[0281] Example 3 In vitro cytolytic activity of the constructs of the present disclosure The in vitro cytolytic activity of M28z-KITv CAR T cells and Mz-KITv CAR T cells against high- and low-MSLN-expressing cells was evaluated. The results are shown in Figures 7A-7B, 8A-8B, and 9. As shown in Figures 7A-7B and 8A-8B, M28z-KITv CAR T cells and Mz-KITv CAR T cells killed high-MSLN tumor cells faster than M28z CAR T cells and Mz CAR T cells; for example, at 4 hours, M28z-KITv CAR T cells killed more tumor cells than M28z CAR T cells, and Mz-KITv CAR T cells killed more tumor cells than Mz CAR T cells (see Figures 7A and 8A), but no significant difference was observed at 18 hours (see Figures 7B and 8B).

[0282] However, for low-MSLN-expressing tumor cells, i.e., A549G cells, M28z-KITv CAR T cells and Mz-KITv CAR T cells showed increased killing ability. At 18 hours, M28z-KITv CAR T cells and Mz-KITv CAR T cells killed more MSLN-low A549G tumor cells than M28z CAR T cells.

[0283] Example 4 PD1 expression and T cell status of constructs after antigen stimulation PD1 expression after antigen stimulation was evaluated. After stimulation with A549GM cells (E:T=3:1) every 4 days, FACS was measured. The results for PD1 expression are shown in Figures 10A and 10B. As shown in Figures 10A and 10B, M28z-KITv CAR T cells and Mz-KITv CAR T cells showed significantly higher CD4 expression than M28z CAR T cells. + T cells (see Figure 10A) and CD8 +Both M28z-KITv CAR T cells and Mz-KITv CAR T cells showed less PD1+ expression after antigen stimulation. The results for T cell status are shown in Figure 11A. As shown in Figure 11A, M28z-KITv CAR T cells and Mz-KITv CAR T cells generated more stem cell-like memory T cells (T SCM cells). SCM The cells have a higher potential to kill tumor cells. M28z-KITv CAR T cells and Mz-KITv CAR T cells secreted higher IFN-γ and TNF-α than M28z and Mz CAR T cells, but less IL-2 (Figure 11B). Thus, the cKIT costimulatory construct demonstrated successful effector cytokine secretion.

[0284] The p-ERK signal of CAR T cells after antigen stimulation was also examined. After co-culture with MGM cells for 5 minutes (E:T=1:2), the p-ERK levels of CD4+ and CD8+ CAR T cells were measured by FACS. Both M28z-KITv and Mz-KITv CD4 and CD8 CAR T cells had stronger p-ERK activity than M28z (Figure 20).

[0285] Example 5 In vivo activity of the constructs of the present disclosure Low-mesothelin-expressing lung cancer cells (A549G) and high-mesothelin-expressing lung cancer cells (A549GM) were used to establish lung tumors in NSG mice (Figure 14C). Mice bearing established low-mesothelin A549G lung tumors were cultured at 1 x 10 5NSG mice were treated with a single dose of M28z, M28z-KITv, and Mz-KITv CAR T cells. Tumor burden in NSG mice was monitored using in vivo BLI. The results are shown in Figures 12A-12D. As shown in Figures 12A-12D, when mice with tumor burdens resulting from cancer cells with low antigen (mesothelin) expression were treated, mice treated with M28z-KITv CAR T cells showed better and longer-lasting tumor regression compared to mice treated with M28z CAR T cells, Mz-KITv CAR T cells, or untransduced T cells (UT). Mouse survival data are shown as Kaplan-Meier survival curves in Figure 14A. As shown in Figure 14A, when mice bearing low antigen (mesothelin)-expressing tumors were treated, the best survival was achieved in mice treated with a single dose of M28z-KITv CAR T cells, e.g., mice treated with M28z-KITv CAR T cells showed extended survival times compared to mice treated with M28z CAR T cells.

[0286] Mice bearing established MSLN-rich A549GM lung tumors were cultured at 1 x 10 5NSG mice were treated with a single dose of M28z, M28z-KITv, and Mz-KITv CAR T cells. Tumor burden in NSG mice was monitored using in vivo BLI. The results are shown in Figures 13A-13D. As shown in Figures 13A-13D, when mice with tumor burdens resulting from cancer cells with high antigen (mesothelin) expression were treated, mice treated with M28z-KITv CAR T cells showed better and longer-lasting tumor eradication. Furthermore, mice treated with Mz-KITv CAR T cells showed long-lasting tumor regression comparable to mice treated with M28z CAR T cells. Mice treated with either mesothelin CAR T cells showed impressive tumor regression compared to mice treated with untransduced (UT) T cells. Mouse survival data are shown as Kaplan-Meier survival curves in Figure 14B. As shown in Figure 14B, when mice bearing high antigen (mesothelin)-expressing tumors were treated, best survival was achieved in mice treated with a single dose of either M28z-KITv or Mz-KITv CAR T cells compared with mice treated with M28z CAR T cells. Median survival times were not reached in mice in all three groups compared with mice treated with untransduced (UT) T cells.

[0287] Example 6 Sensitivity of constructs to tyrosine kinase inhibitors Seven days after stimulation with A549GM cells (E:T=3:1), CAR T were treated with 500nM or 5μM dasatinib (Dasa), 500nM or 5μM ponatinib (Pona), or 100nM or 1μM PKC412 for 72 hours, and then viable cells were measured by FACS. As shown in Figure 15, M28z-KITv CAR T were more sensitive to tyrosine kinase inhibitors than M28z CAR T cells.

[0288] Example 7 In vivo activity of the constructs of the present disclosure Lung tumors were established in NSG mice using A549GM tumor cells, which have high levels of mesothelin (MSLN) expression, or A549G tumor cells, which have low levels of mesothelin (MSLN) expression. Mice were then cultured at 1 x 10 5 Patients were treated with a single dose of M28z, M28z-KITv, or Mz-KITv CAR T cells. Both Mz-KITv and M28z-KITv CAR T cells had higher antitumor efficacy against high-mesothelin-expressing lung cancer cells than M28z CAR T cells (Figures 16A-16C). In low-MSLN A549G lung cancer, M28z-KITv CAR T cells had enhanced antitumor activity compared with M28z CAR T cells, whereas Mz-KITv CAR T cells had lower antitumor activity than M28z CAR T cells (Figures 17A-17C).

[0289] MSLN protein was overexpressed in MSTO cells to generate MG-LM and MGM cells, respectively (Figure 19A). In the pleural mesothelioma tumor model, mesothelioma was established in NSG mice via intrapleural injection of high-MSLN-expressing (MGM) or low-MSLN-expressing (MG-LM) tumor cells. These mice were then injected with 5x10 4 A single dose of P28z, Mz, M28z, M28z-KITv, or Mz-KITv CAR T cells was administered intrathoracically. In high-MSLN-expressing mesotheliomas, Mz-KITv CAR T cells had increased antitumor activity compared with Mz CAR T cells, but no significant difference was detected between M28z-KITv CAR T cells and M28z CAR T cells (Figures 18A and 18B). The CAR T cell dose was intentionally reduced to mimic the low E:T ratio observed in the clinic. The reduced cKIT CAR T cell dose resulted in comparable antitumor efficacy compared with CD28 CAR T cells. This may be due to PDL1 / PD1 pathway-related exhaustion at very low E:T ratios. M28z-KITv CAR T cells also had enhanced antitumor activity against low-MSLN-expressing mesotheliomas (Figure 19B).

[0290] CAR T cells from mice were exposed to high mesothelin-expressing mesothelioma cells and analyzed for PD1 expression. PD1 upregulation was lower at high E:T ratios but similar at low E:T ratios (Figures 21A and 21B).

[0291] Example 8 in vitro nano string analysis MSLN + After 24 hours of co-culture with tumor cells, CD8 + M28z and M28z-KITv CAR T cells were collected for nanostring analysis of CAR T panel genes. 87 of 780 detected genes had significant fold changes (Figure 22A). Pathway score heatmaps showed enriched gene sets for phenotypic and functional T cell traits in M28z-KITv CAR T cells (Figure 22B). Upregulated gene pathways in M28z-KITv CAR T cells are listed in Figure 22C. Expression of type I interferon signaling genes (Figure 23A) and type II interferon signaling genes (Figure 23B) was significantly increased in M28z-KITv CAR T cells. It was significantly increased in CAR T cells.

[0292] Embodiments of the subject matter of the present disclosure From the foregoing description, it is apparent that changes and modifications may be made to the subject matter of the present disclosure to adapt it to various uses and conditions. Such embodiments also fall within the scope of the following claims.

[0293]

[0023] Herein, the reference to a list of elements in any definition of a variable includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. Herein, the reference to an embodiment includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0294] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims

[Claim 1] The invention described in this specification.

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