Immunoresponsive cells expressing dominant negative Fas and uses thereof - Patents.com

Abstract

The present disclosure provides methods and compositions for enhancing immune responses to cancer and pathogens. This relates to cells comprising an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) and a dominant-negative Fas polypeptide. In certain embodiments, the cells are antigen-directed and exhibit enhanced cell persistence and enhanced efficacy of anti-target treatment. In certain embodiments, the dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 738,317, filed Sep. 28, 2018, the contents of which are incorporated herein by reference in their entirety and claim priority thereto. Grant Information

[0002] This invention was made with Government support under Grant Nos. ZIA BC011586 and ZIA BC010763 awarded by the Intramural Research Program of the NCI, Center for Cancer Research of the NIH. The Government has certain rights in this invention. This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on October 16, 2019, is named 072734.0934_SL.txt, and is 42,831 bytes in size. Introduction

[0003] The subject matter of the present disclosure provides a method and composition for enhancing immune response against cancer and pathogens. It relates to an immunoresponsive cell that comprises a dominant negative Fas polypeptide. The immunoresponsive cell can further comprise an antigen recognition receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)). [Background technology]

[0004] Adoptive cellular immunotherapy with genetically engineered autologous or allogeneic T cells has shown evidence of therapeutic efficacy in a range of human cancers, including but not limited to melanoma and various B-cell malignancies. T cells can be engineered to target tumor-associated antigens by the introduction of artificial T cell receptors, termed chimeric antigen receptors (CARs), or genes encoding T cell receptors (TCRs), that convey specificity for antigens expressed by cancer or virus-infected cells. Immunotherapy is a targeted therapy with potential for providing treatment of cancer.

[0005] Adoptive cell transfer (ACT) using genetically engineered T cells has become standard of care for patients with refractory B-cell malignancies, including acute lymphoblastic leukemia in children (1) and aggressive B-cell lymphoma in adults (2). The exceptional efficacy of ACT in hematolymphoid malignancies has been consistently observed across clinical trials, regardless of center, gene vector, or cell composition (3–8). In contrast, responses to adoptive immunotherapy in patients with solid malignancies, which collectively are the leading cause of cancer-related deaths in adults (9), have been relatively modest (10–13). Thus, there remains a need for new strategies to enhance the efficacy of transferred T cells. Summary of the Invention [Means for solving the problem]

[0006] The subject matter of the present disclosure provides a cell (e.g., a T cell, a tumor-infiltrating lymphocyte, or a natural killer (NK) cell) comprising a dominant-negative Fas polypeptide. In certain embodiments, the cell comprises (a) an antigen-recognizing receptor (e.g., a CAR or a TCR) that binds to an antigen, and (b) an exogenous dominant-negative Fas polypeptide. In certain embodiments, the dominant-negative Fas polypeptide comprises at least one modification in a cytoplasmic death domain. In certain embodiments, the at least one modification is selected from the group consisting of a mutation, a deletion, or an insertion. In certain embodiments, the at least one modification is in the cytoplasmic death domain of human Fas. In certain embodiments, the at least one modification in the cytoplasmic death domain prevents binding between the dominant-negative Fas polypeptide and a FADD polypeptide. In certain embodiments, the dominant-negative Fas polypeptide comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence set forth in SEQ ID NO:10. In certain embodiments, a dominant negative Fas polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, a dominant negative Fas polypeptide has the amino acid sequence set forth in SEQ ID NO: 12.

[0007] In certain embodiments, the dominant negative Fas polypeptide comprises a point mutation at position 260 of human Fas having the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the point mutation of human Fas is D260V. In certain embodiments, the dominant negative Fas polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the dominant negative Fas polypeptide has the amino acid sequence set forth in SEQ ID NO: 14.

[0008] In certain embodiments, the exogenous dominant-negative Fas polypeptide enhances cellular persistence of an immunoresponsive cell. In certain embodiments, the exogenous dominant-negative Fas polypeptide reduces apoptosis or anergy of an immunoresponsive cell.

[0009] In certain embodiments, the antigen-recognizing receptor is exogenous or endogenous (e.g., natural antigen specificity from T cells obtained from peripheral blood after in vitro sensitization and / or selection, or from tumor-infiltrating lymphocytes). In certain embodiments, the antigen-recognizing receptor is recombinantly expressed. In certain embodiments, the antigen-recognizing receptor is expressed from a vector.

[0010] In certain embodiments, the exogenous dominant negative Fas polypeptide is expressed from a vector.

[0011] In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a lymphocytic or myeloid lineage cell. In certain embodiments, the cell is selected from the group consisting of T cells, natural killer (NK) cells, B cells, monocytes, and macrophages. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL), a regulatory T cell, or a natural killer T (NKT) cell. In certain embodiments, the immunoresponsive cell is autologous or allogeneic to the intended recipient.

[0012] 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 antigen is a tumor antigen.In certain embodiments, the tumor antigen is 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-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K light chain, KDR, mutant KRAS, mutant PIK3CA, mutant IDH, mutant p53, mutant The tumor antigen is selected from the group consisting of NRAS, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. In certain embodiments, the tumor antigen is CD19.

[0013] In certain embodiments, the antigen is a pathogen-associated antigen. In certain embodiments, the pathogen-associated antigen is a viral antigen present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), or influenza virus.

[0014] In certain embodiments, the antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the antigen recognition receptor is an endogenous TCR that recognizes a pathogen-associated antigen, and the cell is a pathogen-specific T cell. In certain embodiments, the antigen recognition receptor is an endogenous TCR that recognizes a tumor antigen, and the cell is a tumor-specific T cell. In certain embodiments, the antigen recognition 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 CAR further comprises a costimulatory signaling domain. In certain embodiments, at least one costimulatory signaling domain comprises a CD28 polypeptide.

[0015] In certain embodiments, the cell further comprises a suicide gene. In certain embodiments, the suicide gene is herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), or a truncated human epidermal growth factor receptor (EGFRt) polypeptide.

[0016] The subject matter of the present disclosure provides a composition (e.g., pharmaceutical composition) that comprises an effective amount of cells disclosed herein.In certain embodiments, the composition is a pharmaceutical composition that further comprises a pharma- ceutically acceptable carrier.In certain embodiments, the composition is for treating and / or preventing neoplasia and / or pathogen infection.

[0017] The subject matter of the present disclosure provides a method for inducing and / or enhancing an immune response to a target antigen. In certain embodiments, the method comprises administering to a subject an effective amount of a cell disclosed herein or a pharmaceutical composition comprising the same.

[0018] The subject matter of the present disclosure provides a method for reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of cells disclosed herein or a pharmaceutical composition comprising the same. In certain embodiments, the method reduces the number of tumor cells. In certain embodiments, the method reduces tumor size. In certain embodiments, the method eradicates tumor in the subject.

[0019] The subject matter of the present disclosure provides a method for treating and / or preventing neoplasia, or for prolonging the survival of a subject with neoplasia. In certain embodiments, the method comprises administering to the subject an effective amount of a cell or a pharmaceutical composition comprising the same.

[0020] In certain embodiments, the tumor or neoplasm is selected from the group consisting of blood cancer, B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, myeloid leukemia, and myelodysplastic syndrome (MDS). In certain embodiments, the neoplasm is B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin's lymphoma, and the antigen is CD19. In certain embodiments, the neoplasm is selected from solid cancers. The selected solid malignant tumors may include cancers originating from the brain, breast, lung, gastrointestinal tract (including the esophagus, stomach, small intestine, large intestine, and rectum), pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver.

[0021] The subject matter of the present disclosure provides a method for treating hematological cancer in a subject.In certain embodiments, the method comprises administering to the subject an effective amount of T cells, the T cells comprising an antigen-recognizing receptor that binds to an antigen, and an exogenous dominant-negative Fas polypeptide.In certain embodiments, the hematological cancer is selected from the group consisting of B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia and non-Hodgkin's lymphoma, myeloid leukemia, and myelodysplastic syndrome (MDS).

[0022] The subject matter of the present disclosure provides a method for treating solid tumors in a subject.In certain embodiments, the method comprises administering to the subject an effective amount of T cells, the T cells comprising an antigen-recognizing receptor that binds to an antigen, and an exogenous dominant-negative Fas polypeptide.In certain embodiments, the solid tumor is selected from the group consisting of tumors originating from the brain, breast, lung, gastrointestinal tract (including esophagus, stomach, small intestine, large intestine and rectum), pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver.

[0023] The subject matter of the present disclosure provides a method for preventing and / or treating pathogen infection in a subject.In certain embodiments, the method comprises administering to the subject an effective amount of cells or pharmaceutical compositions comprising the cells disclosed herein.In certain embodiments, the pathogen is selected from the group consisting of viruses, bacteria, fungi, parasites and protozoa that can cause disease.

[0024] The subject matter of the present disclosure provides a method for generating antigen-specific cells. In certain embodiments, the method includes introducing into a cell (a) a first nucleic acid sequence encoding an antigen-recognizing receptor that binds to an antigen, and (b) a second nucleic acid sequence encoding an exogenous dominant-negative Fas polypeptide. In certain embodiments, one or both of the first and second nucleic acid sequences are operably linked to a promoter element. In certain embodiments, one or both of the first and second nucleic acid sequences are included in a vector. In certain embodiments, the vector is a retroviral vector.

[0025] The subject of the present disclosure provides a nucleic acid composition comprising: (a) a first nucleic acid sequence encoding an antigen-recognition receptor; and (b) a second nucleic acid sequence encoding an exogenous dominant-negative Fas polypeptide.In certain embodiments, one or both of (a) and (b) are operably linked to a promoter element.In certain embodiments, one or both of the first and second nucleic acid sequences are included in a vector.In certain embodiments, the vector is a retroviral vector.

[0026] The presently disclosed subject matter further provides vectors comprising the nucleic acid compositions disclosed herein.

[0027] The subject matter of the present disclosure provides a kit comprising the cell disclosed herein, the nucleic acid composition disclosed herein, or the vector disclosed herein.In certain embodiments, the kit further comprises a written instruction manual for treating and / or preventing neoplasia and / or pathogen infection. The present invention provides, for example, the following items. (Item 1) (a) an antigen-recognizing receptor that binds to an antigen; and (b) an exogenous dominant-negative Fas polypeptide; Cells containing (Item 2) 2. The cell of item 1, wherein the dominant negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain of human Fas. (Item 3) 3. The cell of item 2, wherein the at least one modification is selected from the group consisting of a mutation, a deletion, and an insertion. (Item 4) 4. The cell of item 3, wherein the mutation is a point mutation. (Item 5) 5. The cell of any one of items 2 to 4, wherein the at least one modification is in the cytoplasmic death domain of human Fas. (Item 6) 6. The cell of any one of items 2 to 5, wherein the at least one modification in the cytoplasmic death domain prevents binding between the dominant negative Fas polypeptide and a FADD polypeptide. (Item 7) 7. The cell according to any one of items 1 to 6, wherein the dominant negative Fas polypeptide comprises a deletion of amino acids 230 to 314 of human Fas having the amino acid sequence shown in SEQ ID NO:10. (Item 8) 8. The cell of item 7, wherein the dominant negative Fas polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:12. (Item 9) The cell of item 8, wherein the dominant negative Fas polypeptide has the amino acid sequence set forth in SEQ ID NO:12. (Item 10) 7. The cell of any one of items 1 to 6, wherein the dominant negative Fas polypeptide comprises a point mutation at position 260 of human Fas having the amino acid sequence set forth in SEQ ID NO: 10. (Item 11) 11. The cell of item 10, wherein the point mutation is D260V. (Item 12) 12. The cell of item 10 or 11, wherein the dominant negative Fas polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:14. (Item 13) 13. The cell of item 12, wherein the dominant negative Fas polypeptide has the amino acid sequence set forth in SEQ ID NO:14. (Item 14) 14. The cell of any one of items 1 to 13, wherein the exogenous dominant-negative Fas polypeptide enhances cellular persistence of an immunoresponsive cell. (Item 15) 15. The cell of any one of items 1 to 14, wherein the exogenous dominant-negative Fas polypeptide reduces apoptosis or anergy of an immunoresponsive cell. (Item 16) 16. The cell of any one of items 1 to 15, wherein the antigen recognition receptor is exogenous or endogenous. (Item 17) 17. The cell of any one of items 1 to 16, wherein the antigen recognition receptor is recombinantly expressed. (Item 18) 18. The cell of any one of items 1 to 17, wherein the antigen recognition receptor is expressed from a vector. (Item 19) 19. The cell of any one of items 1 to 18, wherein the exogenous dominant-negative Fas polypeptide is expressed from a vector. (Item 20) 20. The cell of any one of items 1 to 19, which is an immunoresponsive cell. (Item 21) 21. The cell of any one of items 1 to 20, which is a cell of lymphoid lineage or a cell of myeloid lineage. (Item 22) 22. The cell of any one of items 1 to 21, selected from the group consisting of a T cell, a natural killer (NK) cell, a B cell, a monocyte and a macrophage. (Item 23) 23. The cell of any one of items 1 to 22, which is a T cell. (Item 24) 24. The cell of item 23, wherein the T cell is a cytotoxic T lymphocyte (CTL), a regulatory T cell or a natural killer T (NKT) cell. (Item 25) 25. The cell of any one of paragraphs 1 to 24, which is autologous or allogeneic to the intended recipient. (Item 26) 26. The cell of any one of items 1 to 25, wherein the antigen is a tumor antigen or a pathogen antigen. (Item 27) 27. The cell of any one of items 1 to 26, wherein the antigen is a tumor antigen. (Item 28) The tumor antigen is selected from the group consisting of CD19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K light chain, KDR, mutant KRAS, mutant PIK3CA, mutant IDH, mutant p53, mutant NRAS, 28. The cell of item 27, wherein the cell is selected from the group consisting of LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. (Item 29) 29. The cell of item 28, wherein the antigen is CD19. (Item 30) 30. The cell of any one of items 1 to 29, wherein the antigen is a pathogen-associated antigen. (Item 31) 31. The cell of item 30, wherein the pathogen-associated 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. (Item 32) 32. The cell of any one of items 1 to 31, wherein the antigen recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). (Item 33) 33. The cell of item 32, wherein the antigen recognition receptor is an endogenous TCR that recognizes a pathogen-associated antigen, and the cell is a pathogen-specific T cell. (Item 34) 33. The immunoresponsive cell of claim 32, wherein the antigen-recognizing receptor is an endogenous TCR that recognizes a tumor antigen, and the cell is a tumor-specific T cell. (Item 35) 33. The cell of any one of items 1 to 32, wherein the antigen recognition receptor is a CAR. (Item 36) 36. The cell of claim 35, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. (Item 37) 37. The cell of item 36, wherein the intracellular signaling domain further comprises at least one costimulatory signaling domain. (Item 38) 38. The cell of item 37, wherein the at least one costimulatory signaling domain comprises a CD28 polypeptide. (Item 39) 39. The cell of any one of items 1 to 38, further comprising a suicide gene. (Item 40) 40. The cell of item 39, wherein the suicide gene is herpes simplex virus thymidine kinase (hsv-tk), inducible caspase-9 suicide gene (iCasp-9), or a truncated human epidermal growth factor receptor (EGFRt) polypeptide. (Item 41) 41. A composition comprising an effective amount of the cells according to any one of items 1 to 40. (Item 42) 42. The composition according to item 41, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient. (Item 43) 43. The composition according to item 41 or 42, for treating and / or preventing neoplasia or pathogen infection. (Item 44) 43. A method for inducing and / or enhancing an immune response against a target antigen, comprising administering to a subject an effective amount of a cell according to any one of items 1 to 40 or a composition according to any one of items 42 or 43. (Item 45) 43. A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of a cell according to any one of items 1 to 40 or a composition according to any one of items 42 or 43. (Item 46) 46. ​​The method of claim 45, wherein the number of tumor cells is reduced, the size of the tumor is reduced, and / or the tumor is eradicated in the subject. (Item 47) A method for treating and / or preventing neoplasia, comprising administering to a subject an effective amount of a cell described in any one of items 1 to 40 or a composition described in any one of items 42 or 43. (Item 48) A method for prolonging survival of a subject having a neoplasia, comprising administering to said subject an effective amount of a cell described in any one of items 1 to 40 or a composition described in any one of items 42 or 43. (Item 49) 49. The method of any one of items 45 to 48, wherein the tumor or neoplasm is selected from the group consisting of hematological cancer, B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, myeloid leukemia and myelodysplastic syndrome (MDS). (Item 50) 50. The method of claim 49, wherein the neoplasm is B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia or non-Hodgkin's lymphoma and the antigen is CD19. (Item 51) 1. A method of treating a hematological cancer in a subject in need thereof, comprising administering to the subject an effective amount of a T cell, the T cell comprising an antigen recognition receptor that binds to an antigen, and an exogenous dominant negative Fas polypeptide. (Item 52) 52. The method of claim 51, wherein the hematological cancer is selected from the group consisting of B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, myeloid leukemia and myelodysplastic syndrome (MDS). (Item 53) 1. A method of treating a solid tumor in a subject in need thereof, comprising administering to the subject an effective amount of a T cell, the T cell comprising an antigen recognition receptor that binds to an antigen, and an exogenous dominant-negative Fas polypeptide. (Item 54) 54. The method of item 53, wherein the solid tumor is a tumor of brain, breast, lung, gastrointestinal tract (including esophagus, stomach, small intestine, large intestine and rectum), pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver origin. (Item 55) 41. A method for preventing and / or treating a pathogen infection in a subject, comprising administering to the subject an effective amount of a cell according to any one of items 1 to 40 or a composition according to any one of items 42 or 43. (Item 56) 56. The method of claim 55, wherein the pathogen is selected from the group consisting of a virus, a bacterium, a fungus, a parasite, and a protozoan capable of causing disease. (Item 57) 1. A method for generating an antigen-specific cell, the method comprising introducing into the 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 an exogenous dominant-negative Fas polypeptide. (Item 58) 58. The method of claim 57, wherein one or both of the first and second nucleic acid sequences are operably linked to a promoter element. (Item 59) 59. The method of item 57 or 58, wherein one or both of the first and second nucleic acid sequences are contained in a vector. (Item 60) 60. The method of claim 59, wherein the vector is a retroviral vector. (Item 61) A nucleic acid composition comprising: (a) a first nucleic acid sequence encoding an antigen-recognizing receptor; and (b) a second nucleic acid sequence encoding an exogenous dominant-negative Fas polypeptide. (Item 62) 62. The nucleic acid composition of claim 61, wherein one or both of the first and second nucleic acid sequences are operably linked to a promoter element. (Item 63) 63. The nucleic acid composition according to item 61 or 62, wherein one or both of the first and second nucleic acid sequences are contained in a vector. (Item 64) 64. The nucleic acid composition of item 63, wherein the vector is a retroviral vector. (Item 65) 65. A vector comprising the nucleic acid composition of any one of items 61 to 64. (Item 66) A kit comprising the cell of any one of items 1 to 79, the composition of any one of items 1 to 40, the nucleic acid composition of any one of items 61 to 64, or the vector of item 65. (Item 67) 70. The kit of item 66, further comprising written instructions for treating and / or preventing a neoplasia or pathogen infection.

[0028] The following detailed description may be understood in conjunction with the accompanying drawings, which are given by way of example and without any intent to limit the subject matter of the present disclosure to the specific embodiments described. [Brief description of the drawings]

[0029] [Figure 1-1]Figure 1A-1F: Human tumor microenvironments overexpress the death-inducing ligand FASLG. (A) Cross-cancer analysis of FASLG expression within the microenvironments of 26 different tumor types versus matched normal tissues of origin. RNA sequencing (RNA-seq) data from human cancer and matched normal tissues were extracted from the Cancer Genome Atlas (TCGA) and genotype-tissue expression datasets, analyzed using UCSC Xena, and displayed as RNA-seq normalized by expectation maximization (RSEM) values. Statistical comparison of expression between tumor and normal tissues was performed using Mann-Whitney t-test with Bonferroni correction; ***P<0.001, **P<0.01, *P<0.05. (B) Selected pre-ranked gene set enrichment analysis (GSEA) for all KEGG pathways of genes positively correlated with FASLG expression averaged across 26 TCGA tissue structures. The diameter of the circle reflects the number of genes identified within the GSEA feature set. Nominal P-values ​​and FDR q-values ​​for all displayed GSEAs were <0.001. (C) Pearson correlation of the top 200 correlated genes to FASLG gene expression across 26 human cancer types in the TCGA database. Selected immune-related genes associated with the GSEA feature set listed in panel (B) are identified. (D,E) Representative histograms (D) and summary plots of Fas MFI (E) in phenotypically defined CD8a+ T-cell subsets. Data shown are from peripheral blood T cells from 47 patients and HD. CD8+ T cell subsets in panels (D) and (E) were defined as follows: TN cells, CD8a+CD45RA+CD45RO-CCR7+CD62L+CD27+CD28+Fas-; TCM, CD8a+CD45RO+CD45RA-CCR7+CD62L+; TEM, CD8a+CD45RO+CD45RA-CCR7-CD62L-; TEMRA, CD8a+CD45RA+CCR7-CD62L-.(F) Fraction of TNs among all circulating CD8a+ T cells in age-matched healthy donors (HD; n=39; left) and patients with melanoma (MEL; n=20; center) and diffuse large B-cell lymphoma (DLBCL; n=17; right) at the time of enrollment in an adoptive immunotherapy clinical trial. ***P<0.001, ns=not significant (2-way ANOVA). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0030] [Figure 2-1] Figures 2A-2D show that Fas DNR-engineered mouse T cells prevent FasL-mediated apoptosis. (A) Schematic of physiological Fas signaling and the design of two mouse Fas dominant-negative receptors (DNRs). Retrovirally encoded Fas DNRs were designed to prevent recruitment of Fas-associated protein with death domain (FADD) by either (i) substitution of asparagine for isoleucine residue at position 246 of the death domain (DD; FasI246N) or (ii) truncation of most of the intracellular death domain (FasΔDD). Wild-type Fas (FasWT) and empty vector were used as controls. Receptors were cloned into a bicistronic vector containing a Thy1.1 reporter. EC, extracellular domain; TM, transmembrane domain; T2A, thosea asigna virus 2A self-cleaving peptide. (B) Experimental timeline for stimulation, retroviral transduction, expansion and testing of lz-FasL-mediated apoptosis of WT CD8α+ T cells modified with FasI246N, FasΔDD, FasWT, or empty vector control. (C) Representative FACS plots and (D) summary bar graph showing the frequency of apoptotic Annexin V+ / PI+ transduced T cells at rest and after 6 h exposure to lz-FasL (50 ng mL-1). Results are shown after gating on transduced Thy1.1+ cells. Data shown are representative of six independently performed experiments and are displayed as mean ± SEM with n = 3 per condition. ***P < 0.001, ns = not significant (two-way ANOVA). [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0031] [Figure 3-1]3A-3H are diagrams depicting enhanced survival of Fas DNR engineered T cells in the tumor microenvironment. (A) Experimental outline for the generation and co-injection of congenic WT pmel-1 CD8α+ T cells engineered with FasΔDDDNR (Ly5.1+Thy1.1+) or empty vector control (Ly5.1-Thy1.1+). Transduced T cells were enriched with microbeaded anti-Thy1.1 prior to recombination in an approximately 1:1 mixture, and a total of 8e6 T cells were injected iv into sublethally irradiated (6 Gy) Thy1.1-Ly5.1- mice bearing 10-day established B16 melanoma tumors. Recipient mice received IL-2 by daily ip injection for 3 days, and spleens and tumors were harvested for analysis on day 7. (B) Relative persistence of FasΔDDDNR engineered T cells versus empty vector engineered T cells in recipient mouse spleens and tumors. Results are shown after gating on live CD8α+Thy1.1+ lymphocytes and represent two independent experiments with n=5–8 mice each. ***P<0.001 (unpaired two-tailed Student's t-test). (C) Representative FACS plots and (D) summary bar graphs of T cell viability after overnight culture in cytokine-free medium alone, in the presence of B16 melanoma, or with lz-FasL (50 ng mL-1). T cells were transduced with either FasΔDDDNR or empty vector control without bead enrichment before the start of overnight culture. Data are shown after gating on Thy1.1+ and Thy1.1- lymphocytes. Bar graphs are shown as mean ± SEM and represent four independent experiments with n=3 replicates per condition. (E) Relative persistence of FasΔDDDNR-modified T cells versus empty vector-modified T cells in the spleens and tumors of recipient mice. Results after gating on live CD8α+Thy1.1+ lymphocytes are representative of two independent experiments with n=5-8 mice each. ****P<0.0001, **P<0.01, paired two-tailed Student's t-test. (F) Total number of live Ly5.1+CD8α+Vβ13+ cells transduced with empty or FasΔDD constructs. (G) Relative fold expansion of FasΔDD normalized to empty construct found in spleens on the indicated days.(H) Percentage of live Ly5.1+CD8α+Vβ13+ cells expressing Ki-67 for each condition. Representative plots from two independent experiments. Data are presented as mean ± SEM with n=3 per condition. *P<0.05, Wilcoxon rank sum test. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above.

[0032] [Figure 4-1] Figures 4A-4E show that transfer of Fas DNR modified T cells did not result in acquired autoimmune lymphoproliferative syndrome (ALPS). (A) Representative FACS plots and (B) summary bar graphs of the frequency of CD3+B220+CD4-CD8α- double negative T cells in the spleens of WT mice receiving 6 Gy sublethal irradiation followed by adoptive transfer of 5e5 bead-purified, FasΔDDDNR or empty vector control modified Thy1.1+ pmel-1 T cells. Recipient mice also received IL-2 by daily ip injection for 3 days. Age-matched wild-type and Fas-deficient lpr / lpr mice served as negative and positive controls, respectively. (C) Representative FACS plots and (D) summary scatter plots demonstrating the persistence and surface phenotype of transferred pmel-1 T cells modified with FasΔDDDNR or empty vector control after >6 months. All data shown are representative of 5 independent experiments with n = 5-8 mice per cohort, respectively. ***P < 0.001, *P < 0.05 (one-way ANOVA). (E) Experimental design to analyze the long-term persistence of WT pmel-1 CD8α+ T cells engineered with FasΔDD or empty vector control in B6 mice. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0033] [Figure 5-1]5A-5H. Adoptive transfer of Fas DNR modified T cells enhances antitumor efficacy independent of T cell differentiation status. (A) Experimental design for generation of WT pmel-1 CD8+ T cells modified with FasΔDD, FasI246N or empty vector control. (B) Tumor regression and (C) survival of mice bearing established B16 melanoma tumors at 10 days that were left untreated as control or received 5×105 bead-purified FasΔDD, FasI246N or empty vector control modified Thy1.1+ pmel-1 cells. All treated mice were sublethally irradiated (6 Gy) prior to cell injection followed by ip IL-2 for 3 days. (D) Representative FACS plots demonstrating the purity of sorted CD62L+CD44+Thy1.1+TCM-like pmel-1 T cells modified with Fas DNR or empty vector control prior to injection. (E) Tumor shrinkage and (F) survival of mice bearing established B16 melanoma tumors at 10 days that were either untreated or received 5×105 sort-purified TCM-like Thy1.1+ engineered cells. (G) Tumor shrinkage and (H) survival of mice bearing established B16 melanoma tumors at 10 days that were either untreated or received 5×105 sort-purified TCM-like Thy1.1+ engineered cells. All tumor measurements were performed in a blinded manner by independent investigators. Representative results from two independent experiments are shown as mean ± SEM using n=5–8 mice / cohort. Statistical comparisons were performed using Wilcoxon rank sum test (B,E,G) or log-rank Mantel-Cox test (C,F,H). **P<0.01; *P<0.05. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0034] [Figure 6-1]Figures 6A-6D show that genetic engineering with Fas DNR protects human T cells from FasL-induced apoptosis. (A) Schematic of physiological Fas signaling and the design of two human Fas dominant-negative receptors (DNRs). Retrovirally encoded human Fas DNRs were designed to prevent recruitment of Fas-associated protein with death domain (FADD) by either (i) substitution of valine for the aspartic acid residue at position 260 of the death domain (DD; hFasD260V) or (ii) truncation of most of the human intracellular death domain (hFasΔDD; ΔDD = deletion of aa 230-314 of human Fas). Empty vector was used as a negative control. Receptors were cloned into a bicistronic vector containing a Thy1.1 reporter. EC, extracellular domain; TM, transmembrane domain; T2A, 2A self-cleaving peptide derived from Thosea asigna virus 2A. (B) Experimental timeline for stimulation, retroviral transduction, expansion and testing of lz-FasL-mediated apoptosis of human CD8+ T cells derived from peripheral blood mononuclear cells (PBMCs) modified with FasD244V, FasΔDD, or empty vector control. (C) Representative FACS plots and (D) summary graph showing the frequency of apoptotic Annexin V+ T cells at rest and after 6 h exposure to titrating concentrations of lz-FasL. Results are shown after gating on transduced (Thy1.1+) or non-transduced (Thy1.1-) T cells. Data are shown as mean ± SEM with n=3 per indicated condition and represent three independent experiments. *P<0.05, ns=not significant (Wilcoxon rank sum test). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0035] [Figure 7-1]7A-7D are diagrams depicting the design and expression of retrovirally encoded mouse Fas DNR constructs and controls in mouse CD8+ T cells. (A) Schematic of the design of retroviral constructs encoding mouse wild-type (WT) Fas, or mutant forms of Fas impaired in their ability to bind intracellular adaptor molecules associated with Fas via the death domain. WT Fas, Fas with an asparagine replacing the isoleucine at position 246 (FasI246N), or Fas with a truncation of the intracellular death domain (FasΔDD) were cloned into the MSGV1 expression vector before the T2A cleavage site and the Thy1.1 reporter gene. An empty vector containing only the Thy1.1 reporter gene (empty) was used as a negative control. (B) Representative FACS plots and summary bar graphs of (C) Thy1.1 and (D) Fas expression 4 days after retroviral transduction of Fas-deficient lpr / lpr or WT CD8α+ T cells. The percentage of gated Thy1.1+ or Fas+ cells is shown in black, and the MFI of Thy1.1+ or Fas+ cells is shown in red in the flow plots. Data in (C) and (D) are presented as mean ± SEM with n=3 per condition and represent 12 independent experiments. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0036] [Figure 8-1]8A-8D: Fas DNR prevents lz-FasL induced AKT activation and T cell differentiation. (A, B) Representative FACS histograms (top) and summary plots (bottom) of the dose-response relationship between lz-FasL exposure and (A) phospho-AKTS473 and (B) phospho-S6S235 / 236 in CD8α+ T cells transduced with FasI246N, FasΔDD or empty vector control. Results shown after 6 days of activation, retroviral transduction and expansion in the continuous presence of the indicated concentrations of lz-FasL. (C) Representative FACS plots of T cell differentiation (top) and intracellular IFNγ / IL-2 production (bottom) after 11 days when CD8α+ T cells were transduced with FasI246N, FasΔDD or empty vector control in the absence of exogenous FasL. Intracellular cytokine staining was measured after approximately 5 h incubation with PMA / ionomycin in brefeldin A and monensin. (D) Memory T cell subset composition of CD8α+ T cells after 11 days of activation, transduction and expansion in culture. Graphs are shown as mean ± SEM with n=3 per condition and represent three (A, B) and five (C, D) independent experiments. *P<0.05 (Wilcoxon rank sum test). [Figure 8-2] Same as above.

[0037] [Figure 9-1]9A-9E depict the effect of Fas DNR and anti-CD19 CAR modified T cell treatment in a mouse model of leukemia. (A) Experimental design for treatment with syngeneic T cells co-transduced with anti-CD19 CAR and either FasΔDD or empty vector control in a mouse leukemia model. All treated mice were sublethally irradiated (5 Gy) prior to cell injection followed by ip IL-2 for 3 days. (B) Representative FACS plots demonstrating the efficiency of co-transduction and (C) purity of sorted Thy1.1+ T cells modified with anti-CD19 CAR and either FasΔDD or empty vector control. (D) Survival of mice bearing established E2a:PBX pre-B ALL tumors at 10 days that were left untreated as a control or received a high CAR T cell dose (5.5×105) of sorted-purified Thy1.1+ T cells modified with an anti-CD19 CAR and either FasΔDD or an empty vector control. (E) Survival of mice bearing established E2a:PBX pre-B ALL tumors at 10 days that were left untreated or received a low CAR T cell dose (1.8×105) of sorted-purified Thy1.1+ T cells modified with an anti-CD19 CAR and either FasΔDD or an empty vector control. All tumor measurements were performed in a blinded manner by an independent investigator. [Figure 9-2] Same as above.

[0038] [Figure 10-1]Figures 10A-10G show that expression of Fas DNR enhances anti-apoptotic function and in vivo persistence in the anti-CD19 CAR model. (A) Representative flow plots and (B) summary data of dual transduction of B6 CD8α+ T cells with retroviral constructs encoding anti-CD19 CAR and empty or Fas DNR. Analysis was performed 11 days after Thy1.1 bead enrichment on day 6. (C) Summary bar graphs of relative T cell viability (relative to FasΔDD) after overnight culture with cytokine-free medium alone, lz-FasL (100 ng ml-1), 2 μg ml-1 of anti-CD3 and anti-CD28 or E2a-PBX, respectively. Data shown after gating on Thy1.1+ lymphocytes are representative of three independently performed experiments and are displayed as mean ± SEM with n = 3 per condition. *P<0.05, ****P<0.0001, two-way ANOVA. (D) Experimental outline for the generation and infusion of WT CD8α+ T cells engineered to express anti-CD19 CAR together with FasΔDDDNR or empty vector control. Transduced T cells were enriched with Thy1.1 beads prior to injection, and T cells were infused iv into sublethally irradiated (5 Gy) mice harboring 4-day established E2a-PBX leukemia. Spleens and BM were collected for analysis on day 14. co-Td, co-transduction. (E) Summary data for the number of live CD8α+Thy1.1+ lymphocytes in the spleen and BM of recipient mice. (F) Summary data for the frequency of E2a-PBX leukemia in the BM of recipient mice. Results in E and F represent two independent experiments with n=3–5 mice each. *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA corrected for Tukey's multiple comparisons. (G) Survival of mice bearing established E2a-PBX leukemia at 4 days that were untreated or received 3x105 (left) or 2x105 (right) anti-CD19 CAR+Thy1.1+ modified cells. Representative results from 4 independent experiments are shown as mean ± SEM using n=5 mice / cohort. Statistical comparisons were performed using the log-rank Mantel-Cox test; *P<0.05 **P<0.01. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above.

[0039] [Figure 11] Figure 11 shows that Fas DNR can protect non-transduced cells from FasL-mediated apoptosis. Summary bar graph showing the relative frequency of cell viability of non-transduced and transduced T cells 20 hours after exposure to lz-FasL (100 ng mL-1). Results shown after gating on live CD8α+ lymphocytes and viability shown relative to medium for each transduction condition. Data shown are representative of three independent experiments and are displayed as mean ± SEM with n = 3 per condition. ****P < 0.0001, ns = not significant (one-way ANOVA corrected for Tukey's multiple comparisons).

[0040] [Figure 12-1] 12A-12D show that expression of FasI246N in T cells does not cause reversion to WT Fas. (A) Experimental timeline for stimulation, retroviral transduction, and analysis of WT CD8α+ T cells modified with FasWT or FasI246N. (B) Representative FACS plots of Thy1.1 expression on days 6 and 12 for cells transduced with FasWT or FasI246N. (C) Experimental timeline for stimulation, transduction, Thy1.1 enrichment, and sequencing of WT CD8α+ T cells modified with FasWT or FasI246N. (D) Representative sequencing data showing that WT Fas maintains the ACT sequence encoding an isoleucine at amino acid position 246, whereas the FasI246N sequence is AAC encoding an asparagine at amino acid position 246 of the introduced Fas DNR construct. [Figure 12-2] Same as above.

[0041] [Figure 13]Figure 13 depicts IFNγ upregulating FasL on the surface of B16 tumor cells. B16 cells were treated with vehicle (PBS) or IFNγ (100 ng mL-1) for 24 hours and then analyzed for surface expression of MHC class I (H-2Db; left panel) or FasL (right panel) by flow cytometry.

[0042] [Figure 14] Figure 14: Fas DNR engineered T cells prevent apoptosis from various stimuli. Summary bar graph showing relative frequency of cell viability of transduced T cells 20 hours after exposure to lz-FasL (100 ng mL-1). Results are shown after gating on Thy1.1+ cells and viability is shown relative to FasΔDD. Data shown are representative of 10 independently performed experiments and are displayed as mean ± SEM with n = 3 per condition. *P<0.05 **P<0.01 ****P<0.0001, ns = not significant (one-way ANOVA corrected for Tukey's multiple comparisons).

[0043] [Figure 15-1]Figures 15A-15H show that Fas DNR expression does not induce lymphoproliferation in ALPS-susceptible MRL strains. (A) Schematic diagram comparing the onset of lymphoproliferation (top) in 6-9 month old C57BL / 6 B6-lpr mice with 3-4 month old MRL-lpr strains. (B) Experimental design to analyze the long-term persistence of CD8α+ T cells expressing WT anti-CD19 CAR modified with FasΔDD or empty vector control in WT MRL-Mp mice. A total of 3x106 anti-CD19 CAR+CD8α+ T cells were injected iv into sublethally irradiated (6Gy XRT) mice. Recipient mice received IL-2 by daily ip injection for 3 days and spleens were collected for analysis after 93 days. (C) Summary of spleen weights in recipient mice compared to age-matched wild-type and Fas-deficient B6-lpr mice (negative and positive controls, respectively). (D,E) Representative FACS plots (D) and (E) summary bar graphs of the frequency of CD3+B220+ double negative lymphocytes in the spleens of recipient and control mice. (F) Summary bar graphs of antinuclear antibody (ANA) Ig (top) and anti-dsDNA Ig (bottom) levels measured by ELISA. (G,H) Summary bar graphs demonstrating the persistence (G) and surface phenotype (H) of transferred Thy1.1+ T cells modified with FasΔDDDNR or empty vector control. n=27 mice per cohort. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns=not significant (one-way ANOVA corrected for Tukey's multiple comparisons). [Figure 15-2] Same as above. [Figure 15-3] Same as above.

[0044] [Figure 16]16A-16B: Adoptively transferred T cells modified with Fas DNR do not induce inflammatory infiltrates in the lungs of ALPS-susceptible MRL host mice. (A) Representative H&E stained macrophages and (B) summary graphs demonstrating the intensity of inflammatory mononuclear cell infiltrates in the lungs of treated mice. Arrows and stars indicate areas of dense perivascular and peribronchiolar mononuclear inflammatory infiltrates, respectively. Scale bar = 300 μm. All images were scored in a blinded manner by an interpreting pathologist. ***P<0.001, ns=not significant (one-way ANOVA corrected for Tukey's multiple comparisons).

[0045] [Figure 17-1] 17A-17E show the simultaneous genetic engineering of primary human T cells with a Fas dominant-negative receptor (ΔDD), an antigen-specific TCR (NY-ESO-1, 1G4) and a tractable suicide switch (truncated EGFR). (A) Design of the human retroviral constructs used in these experiments. (B) Schematic of primary human T cells co-modified with TCR and FasDNR. (C) Co-expression of human FasDNR and tEGFR suicide switch. (D) Antigen-specific cytokine production and (E) response to lz-FasL. [Figure 17-2] Same as above.

[0046] [Figure 18-1] 18A-18D are diagrams depicting the simultaneous genetic engineering of primary human T cells with a Fas dominant negative receptor (ΔDD), an antigen-specific CAR (anti-CD19, 28z) and a tractable suicide switch (truncated EGFR). (A) Design of the human retroviral constructs used in these experiments. (B) Schematic of primary human T cells co-modified with CAR and FasDNR. (C) Time-dependent induction of apoptosis in human T cells modified with tEGFR alone or in combination with hFasDNR after exposure to lz-FasL. (D) Antigen-specific cytokine release and degranulation in human T cells modified with anti-CD19 CAR alone or in combination with hFasDNR. [Figure 18-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description of the Invention The subject matter of the present disclosure provides cells, including genetically modified immunoresponsive cells (e.g., T cells or NK cells) that contain a dominant-negative Fas polypeptide. In certain embodiments, the immunoresponsive cells further comprise an antigen-recognition receptor (e.g., TCR or CAR). The subject matter of the present disclosure also provides methods of using such cells to induce and / or enhance an immune response to a target antigen, and / or methods of treating and / or preventing a neoplasm, pathogen infection, or other disease / disorder (e.g., a disease / disorder in which an increased antigen-specific immune response is desirable). The subject matter of the present disclosure is based, at least in part, on the discovery that a dominant-negative Fas polypeptide enhances cell persistence, prevents activation that induces cell death, prevents FasL-induced cell death, and / or improves the antitumor effect of an immunoresponsive cell. 1.Definition

[0048] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art.The following references provide those skilled in the art with the general definitions of many 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 below, unless otherwise specified.

[0049] As used herein, the term "about" or "approximately" refers to within an acceptable error range for a particular value as determined by a person skilled in the art, depending in part on the method by which the particular 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 in the art. Alternatively, "about" can mean within 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, e.g., within 5-fold or 2-fold of a value.

[0050] By "immunoresponsive cell" is meant a cell that functions in an immune response, or its ancestors or progeny.

[0051] "Activating an immunoresponsive cell" refers to the induction of signal transduction or change in protein expression in a cell that results in the initiation of an immune response. For example, when CD3 chain clusters respond to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade occurs. In certain embodiments, when endogenous TCR or exogenous CAR binds to an antigen, the formation of an immune synapse occurs, including the clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the bound receptor. This clustering of membrane-bound signal transduction molecules allows the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates the activation pathway of T cells, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce the global gene expression of T cells, which increases IL-2 production for the proliferation and expression of key regulators of T cell proteins to initiate a T cell-mediated immune response.

[0052] "Stimulating immune responsive cells" refers to signals that result in strong and sustained immune responses. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS. Receiving multiple stimulatory signals may be important to boost strong and long-lasting T cell-mediated immune responses. T cells can be quickly inhibited and become unresponsive to antigens. Although the effects of these costimulatory signaling signals may vary, they generally result in increased gene expression to generate long-lived, proliferative and anti-apoptotic T cells that respond strongly to antigens for complete and sustained eradication.

[0053] The term "antigen recognition receptor," as used herein, refers to a receptor that can activate an immune cell or an immunoresponsive cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen recognition receptors include natural or endogenous T cell receptors ("TCRs"), and chimeric antigen receptors ("CARs").

[0054] 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, may be cleared more rapidly from circulation and have less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). As used herein, antibodies include complete natural antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein, V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (herein referred to 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 and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).H and V L is composed of three CDRs and four FRs arranged from amino to 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 may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

[0055] As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody, which is the hypervariable region of the heavy and light chains of immunoglobulins. 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 and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the antibody to bind to antigen or epitope. In certain embodiments, CDR regions are described using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0056] As used herein, the term "single chain variable fragment" or "scFv" refers to a V H ::V L Covalently linked immunoglobulin heavy chains (V H ) and light chain (V L ) is a fusion protein of the variable region of Hand V L are either directly joined or joined by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids), and V H The N-terminus of L and the C-terminus of V H The C-terminus of L The linker is usually rich in glycine for flexibility and serine or threonine for solubility. 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 synthesized by the synthesis of single-chain Fv polypeptides, as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H Coding sequence and V LIt can be expressed from a nucleic acid containing the coding sequence. See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0057] 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 binding site and the antigenic determinant, the size of the area of ​​contact 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 the formation of a reversible complex. Methods for calculating the affinity of an antibody to 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).

[0058] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule that comprises an extracellular antigen-binding domain fused to an intracellular signaling domain that can activate or stimulate an immunoresponsive cell and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be derived from the fusion of the variable heavy and light chain regions of an antibody. Alternatively, or in addition, the scFv can be derived from a Fab (instead of being derived from an antibody, e.g., obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain, which is then fused to an intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for an antigen.

[0059] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule that encodes a polypeptide of interest (e.g., a dominant negative Fas polypeptide) or a fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical to an endogenous nucleic acid sequence, but may exhibit substantial identity. A polynucleotide having "substantial identity" or "substantial homology" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0060] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of the CAR of the present disclosure (e.g., the extracellular antigen-binding domain of the CAR) that contains the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv 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. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid in the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can 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 non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, conservative substitutions 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 certain embodiments, one or more amino acid residues within or outside the CDR regions can be replaced with other amino acid residues from the same group, and the altered antibodies can be tested for retained function (i.e., the functions set forth in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 residues outside the CDR regions or within the specified sequences of the CDR regions are altered.

[0061] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences.The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % homology=number of identical positions / total number of positions×100).Comparing sequences between two sequences and determining the percentage of identity can be achieved using a mathematical algorithm.

[0062] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0063] Additionally or alternatively, the amino acid sequences of the subject matter of the present disclosure 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 using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the sequences identified herein. 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 (e.g., XBLAST and NBLAST) can be used.

[0064] Furthermore, sequence identity can be measured by using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications.

[0065] "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.

[0066] An exemplary approach to determining the degree of identity may use the BLAST program, with a probability score between e-3 and e-100 indicating closely related sequences.

[0067] "Analog" refers to a structurally related polypeptide or nucleic acid molecule that has a function of a reference polypeptide or nucleic acid molecule.

[0068] 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.

[0069] The term "constitutive expression" or "constitutively expressed" as used herein refers to expression or being expressed under all physiological conditions.

[0070] By "disease" is meant any condition, disease or disorder that damages or interferes with the normal function of a cell, tissue or organ, such as neoplasia and pathogenic infection of a cell.

[0071] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect beneficial or desired clinical outcomes 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 of ordinary skill in the art. Several factors are typically considered when determining the appropriate dosage to achieve an effective amount. 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 immunoresponsive cells being administered.

[0072] By "enhancing tolerance" is meant preventing the activity of autoreactive or immunoresponsive cells that target the transplanted organ or tissue.

[0073] By "endogenous" is meant a nucleic acid molecule or a polypeptide that is normally expressed in a cell or tissue.

[0074] "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 foreign, heterologous, overexpressed nucleic acid molecules and polypeptides. "Exogenous" nucleic acid refers to a nucleic acid that is not present in a natural wild-type cell, e.g., an exogenous nucleic acid may vary from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or different sequence relative to its natural endogenous counterpart, may be introduced by genetic engineering into the cell itself or its precursor cell, and may be linked to alternative control sequences, such as non-native promoters or secretion sequences, as appropriate.

[0075] 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 the cell or in a sample obtained from the 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.

[0076] By "modulate" is meant 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%.

[0077] By "increase" is meant a positive alteration of at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or higher.

[0078] By "reduce" is meant to negatively alter by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0079] By "isolated cell" is meant a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0080] The terms "isolated," "purified," or "biologically pure" refer to a material that is free, to varying degrees, from components that normally accompany it as found in its native state. "Isolate" refers to a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not significantly affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may refer to a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications may give rise to different isolated proteins that can be purified separately.

[0081] 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.

[0082] "Linker," as used herein, is intended to mean a functional group (e.g., a chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids such that they are connected to one another. As used herein, a "peptide linker" is a peptide linker that couples two proteins together (e.g., V H and V L The linker refers to one or more amino acids used to couple (connect) domains. In certain embodiments, the linker comprises the sequence set forth in GGGGSGGGSGGGGGS [SEQ ID NO: 1].

[0083] "Neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that would not induce proliferation of normal cells or cause their cessation. Neoplasia can affect a variety of 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 tube, 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. Neoplasia includes cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0084] By "receptor" is meant a polypeptide or portion thereof present on a cell membrane that selectively binds to one or more ligands.

[0085] "Recognize" refers to selective binding to a target. T cells that recognize a tumor can express a receptor (e.g., TCR or CAR) that binds to a tumor antigen.

[0086] "Reference" or "control" refers to a standard for comparison. For example, the level of scFv-antigen binding by a cell expressing a CAR and an scFv can be compared to the level of scFv-antigen binding in a corresponding cell expressing a CAR alone.

[0087] By "secreted" is meant a polypeptide that is released from the cell via the secretory pathway, through the endoplasmic reticulum, the Golgi apparatus, and as vesicles that transiently fuse with the cell plasma membrane, releasing the protein outside the cell.

[0088] By "signal sequence" or "leader sequence" is meant a peptide sequence (e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway. Exemplary leader sequences include, but are not limited to, the IL-2 signal sequence: MYRMQLLSCIALSLALVTNS [SEQ ID NO:2] (human), MYSMQLASCVTLTLVLLVNS [SEQ ID NO:3] (mouse); kappa leader sequence: METPAQLLFLLLLWLPDTTG [SEQ ID NO:4] (human), METDTLLLWVLLLWVPGSTG [SEQ ID NO:5] (mouse); CD8 leader sequence: MALPVTALLLPLALLLHAARP [SEQ ID NO:6] (human); truncated human CD8 signal peptide: MALPVTALLLPLALLLHA [SEQ ID NO:7] (human); albumin signal sequence: MKWVTFISLLFSSAYS [SEQ ID NO:8] (human); and prolactin signal sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO:9] (human). "Soluble" refers to a polypeptide that is freely diffusible (e.g., not membrane bound) in an aqueous environment.

[0089] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds a biological molecule of interest (e.g., a polypeptide) but does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the present disclosure.

[0090] The term "tumor antigen" as used herein refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-IS neoplastic cells. In certain embodiments, tumor antigens include any polypeptide expressed by tumors that can activate or induce an immune response via antigen recognition receptors (e.g., CD19, MUC-16) or suppress an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0091] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc.

[0092] As used herein, "treatment" refers to clinical intervention that seeks to change the disease course of the individual or cell being treated, and can be performed for prevention or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can prevent the deterioration caused by the disorder in a subject who has been affected or diagnosed, or a subject who is suspected of having the disorder, but treatment can also prevent the onset of the disorder or symptoms of the disorder in a subject who is at risk of the disorder or a subject who is suspected of having the disorder.

[0093] An "individual" or "subject" herein is a vertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, livestock, sport animals, rodents, and pets. 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 that is immunocompromised. The subject is highly vulnerable to opportunistic infections, which are infectious diseases 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.

[0094] Other aspects of the presently disclosed subject matter are described in the following disclosure, which are within the scope of the presently disclosed subject matter. 2. Dominant negative Fas polypeptide

[0095] Fas cell surface death receptor (Fas) is also known as APT1, CD95, FAS1, APO-1, FASTM, ALPS1A, TNFRSF6. GenBank ID: 355 (human), 14102 (mouse), 246097 (rat), 282488 (cow), 486469 (dog). Protein products of Fas include, but are not limited to, NCBI reference sequences NP_000034.1, NP_001307548.1, NP_690610.1 and NP_690611.1.

[0096] Fas is a member of the TNF receptor superfamily and contains a death domain. It is involved in the regulation of programmed cell death and has been implicated in the pathogenesis of various malignancies and diseases of the immune system. The interaction of Fas with its ligands allows the formation of a death-inducing signaling complex with other components, such as Fas-associated protein and death domain (FADD), which can induce programmed cell death.

[0097] In certain embodiments, the Fas polypeptide is a human Fas polypeptide. In certain embodiments, the human Fas polypeptide comprises or has the amino acid sequence of NCBI Reference Number: NP_000034.1 (SEQ ID NO: 10) provided below. In certain embodiments, the human Fas polypeptide comprises or has 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 99%, or at least about 100% homologous or identical to the sequence shown in SEQ ID NO: 10. [ka]

[0098] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:10 is shown in SEQ ID NO:11, provided below.

[0099]

[0100] In certain embodiments, the term "dominant negative Fas polypeptide" refers to a dominant negative form of Fas polypeptide that is a gene product of a dominant negative mutation of the Fas gene. In certain embodiments, a dominant negative mutation (also called an "antimorph mutation") has an altered gene product that acts antagonistically to the wild-type allele. In certain embodiments, a dominant negative Fas polypeptide adversely affects the normal wild-type Fas polypeptide in the same cell. In certain embodiments, a dominant negative Fas polypeptide interacts with the wild-type Fas polypeptide but blocks its signaling to downstream molecules, such as FADD.

[0101] In certain non-limiting embodiments, the dominant negative Fas polypeptide comprises a heterologous signal peptide, such as the IL-2 signal peptide, the kappa leader sequence, the CD8 leader sequence, or a peptide having essentially equivalent activity.

[0102] In certain embodiments, the dominant negative Fas polypeptide comprises at least one modification in the intracellular domain. In certain embodiments, the at least one modification prevents binding of Fas to the FADD polypeptide. In certain embodiments, the at least one modification is in the death domain. In certain embodiments, the at least one modification is in about amino acids 200 to about 320 of SEQ ID NO: 10. In certain embodiments, the at least one modification is in about amino acids 200 to about 319 of SEQ ID NO: 10. In certain embodiments, the at least one modification is in about amino acids 202 to about 319 of SEQ ID NO: 10. In certain embodiments, the at least one modification is in about amino acids 226 to about 319 of SEQ ID NO: 10. The death domain of the Fas protein is disclosed in Tartaglia LA et al. Cell. (1993);74(5):845-53; Itoh and Nagata. J Biol Chem. (1993);268(15):10932; Boldin MP et al. J Biol Chem. (1995);270(14):7795-8; and Huang B et al. Nature (1996);384(6610):638-41, all of which are incorporated by reference herein.

[0103] In certain embodiments, the modification is selected from the group consisting of a mutation, a deletion, and an insertion, hi certain embodiments, the mutation is a point mutation.

[0104] In certain embodiments, the modification is a deletion. In certain embodiments, the dominant negative Fas polypeptide comprises a partial or complete deletion of the death domain. In certain embodiments, the dominant negative Fas polypeptide comprises or has a deletion of amino acid residues 230-314 of a human wild-type Fas polypeptide (e.g., having the amino acid sequence set forth in SEQ ID NO: 10). In certain embodiments, a dominant negative Fas polypeptide having a deletion of amino acid residues 230-314 of a human wild-type Fas polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 is referred to as "hFas ΔDD hFas ΔDD has the amino acid sequence shown in SEQ ID NO: 12. SEQ ID NO: 12 is provided below.

[0105] MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLLKDITSDSENSNFRNEIQSLV (SEQ ID NO: 12)

[0106] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:12 is shown in SEQ ID NO:13, provided below.

[0107] ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTATCGTCCAAAAGTGTTAATGCCCAAGTGACTGACATCAACTCCAAGGGATTGGAATTGAGGAAGACTGTTACTACAGTTGAGACTCAGAACTTGGAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAGGTGAAAGGAAAGCTAGGGACTGCACAGTCAATGGGGATGAACCAGACTGCGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCTTCCAAATGCAGAAGATGTAGATTGTGTGATGAAGGACATGGCTTAGAAGTGGAAATAAACTGCACCCGGACCCAGAATACCAAGTGCAGATGTAAACCAAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACCAAATGTGAACATGGAATCATCAAGGAATGCACACTCACCAGCAACACCAAGTGCAAAGAGGAAGGTTCCAGATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTTTGGGTGAAGAGAAAGGAAGTACAGAAAACATGCAGAAAGCACAGAAAGGAAAACCAAGGTTCTCATGAATCTCCAACCTTAAATCCTGAAACAGTGGCAATAAATTTATCTGATGTTGACTTGCTCAAGGACATTACTAGTGACTCAGAAAATTCAAACTTCAGAAATGAAATCCAAAGCTTGGTC(SEQ ID NO: 13)

[0108] In certain embodiments, a dominant negative Fas polypeptide comprises or has 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 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, a dominant negative Fas polypeptide having 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 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12 comprises or has a deletion of amino acid residues 230-314 of a human Fas polypeptide (e.g., one having the amino acid sequence set forth in SEQ ID NO: 10).

[0109] In certain embodiments, the modification is a point mutation. In certain embodiments, the dominant-negative Fas polypeptide comprises or has a point mutation at position 260 of a human Fas polypeptide (e.g., having the amino acid sequence set forth in SEQ ID NO: 10). In certain embodiments, the point mutation is D260V. In certain embodiments, a dominant-negative Fas polypeptide having the point mutation D260V of a human wild-type Fas polypeptide is referred to as "hFas D260V " and hFas D260V has the amino acid sequence shown in SEQ ID NO: 14. SEQ ID NO: 14 is provided below.

[0110] MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIVEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO: 14)

[0111] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:14 is shown in SEQ ID NO:15, provided below.

[0112]

[0113] In certain embodiments, a dominant negative Fas polypeptide comprises or has 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 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, a dominant negative Fas polypeptide having 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 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 14 comprises or has the point mutation D260V of a human Fas polypeptide (e.g., one having the amino acid sequence set forth in SEQ ID NO: 10).

[0114] In certain non-limiting embodiments, the dominant negative Fas polypeptide comprises a heterologous signal peptide, such as the IL-2 signal peptide, the kappa leader sequence, the CD8 leader sequence, or a peptide having essentially equivalent activity. 3. Antigen Recognition Receptor

[0115] The present disclosure provides an antigen-recognizing receptor that binds to 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).The antigen-recognizing receptor can bind to tumor antigen or pathogen antigen. 3.1. Antigen

[0116] In certain embodiments, the antigen recognition receptor binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, cancer proteins. Antigens can be expressed as peptides or as intact proteins or portions thereof. The intact proteins or portions thereof can be natural or mutagenized. Non-limiting examples of tumor antigens include:

[0117] 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-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K light chain, KDR, mutant KRAS (including but not limited to G12V, G12D, G12C), mutant PIK3CA (including but not limited to E52K, E545K, H1047R, H1047L), mutant IDH (including but not limited to R132H), mutant p53 (R175H, Y220C, G245D, G245S), , R248L, R248Q, R248W, R249S, R273C, R273L, R273H and R282W), mutant NRAS (including but not limited to Q61K), LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. In certain embodiments, the tumor antigen is CD19.

[0118] In certain embodiments, the antigen recognition receptor binds to a human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16, provided below.

[0119] PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK ​​[SEQ ID NO: 16]

[0120] In certain embodiments, the antigen recognition receptor binds to the extracellular domain of the human CD19 protein.

[0121] In certain embodiments, the antigen recognition receptor binds to a pathogen antigen, for use in the treatment and / or prevention of, for example, a pathogen infection, for example, in an immunocompromised subject. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites and protozoa that can cause disease.

[0122] Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or HTLV-III / LAV, or HIV-III) and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (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, Bungavirus, Phlebovirus and Nairavirus) viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B viruses); Parvovirida (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes viruses; Poxviridae (smallpox viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever viruses); as well as unclassified viruses (e.g., the etiologic agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), etiologic agents of non-A, non-B hepatitis (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e., hepatitis C);Norwalk and related viruses, and Astroviruses), human papillomaviruses (i.e., HPV), JC virus, Epstein-Barr virus, Merkel cell polyomavirus;

[0123] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species.Specific examples of infectious bacteria include Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (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 sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Clostridium difficile, and Actinomyces israelli.

[0124] 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. 3.2.T cell receptor (TCR)

[0125] 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. A TCR is found on the surface of a T cell and is responsible for recognizing antigens as peptides that bind to major histocompatibility complex (MHC) molecules. In certain embodiments, a TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, a TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0126] 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 and continues to a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity determining regions (CDRs).

[0127] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε and CD247ζ / ζ or ζ / η. When the TCR complex engages with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0128] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the TCR recognizes a viral antigen. In certain embodiments, the TCR is expressed in a virus-specific T cell. In certain embodiments, the virus-specific T cell is derived from an individual's immunity to a viral infection, for example, BK virus, human herpesvirus 6, Epstein-Barr virus (EBV), cytomegalovirus or adenovirus. In certain embodiments, the virus-specific T cell is the T cell disclosed in Leen et al., Blood, Vol. 121, No. 26, 2013; Barker et al., Blood, Vol. 116, No. 23, 2010; Tzannou et al., Journal of Clinical Oncology, Vol. 35, No. 31, 2017; or Bollard et al., Blood, Vol. 32, No. 8, 2014, each of which is incorporated by reference in its entirety. In certain embodiments, TCR recognizes tumor antigen.In certain embodiments, TCR is expressed in tumor-specific T cells.In certain embodiments, tumor-specific T cells are tumor-infiltrating T cells that are generated by culturing T cells with tumor, for example, melanoma or epithelial cancer explants.In certain embodiments, tumor-specific T cells are the T cells disclosed in Stevanovic et al, Science, 356, 200-205, 2017;Dudley et al. Journal of Immunotherapy, 26(4): 332-342, 2003;or Goff et al, Journal of Clinical Oncology, Vol. 34, No. 20, 2016, each of which is incorporated by reference in its entirety.

[0129] 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 by at least one amino acid residue from the naturally occurring TCR. In certain embodiments, a non-naturally occurring TCR has 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 altered from a naturally occurring TCR. Chimeric antigen receptors (CARs)

[0130] In certain embodiments, the antigen recognition receptor is CAR.CAR is an engineered receptor that grafts or gives immune effector cells or immune responsive cells the specificity of interest.CAR can be used to graft the specificity of monoclonal antibodies onto T cells, and the transfer of their coding sequences is facilitated by retroviral vectors.

[0131] 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 can provide de novo antigen recognition and bind to CD4 through their CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.+ T cells and CD8 + "Second generation" CARs can induce both activation of T cells and activation of 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 (CD3zeta). "Third generation" CARs include those that provide multiple costimulation (e.g., CD28 and 4-1BB) and activation (CD3zeta). In certain embodiments, the antigen recognition receptor is a first generation CAR.

[0132] In certain non-limiting embodiments, the extracellular antigen binding domain of the CAR (e.g., embodied in an scFv or analog thereof) is about 2×10 -7 M or less dissociation constant (K d In certain embodiments, K d is about 2 x 10 -7 M or less, approximately 1 x 10 -7 M or less, approx. 9 x 10 -8 M or less, approximately 1 x 10 -8 M or less, approx. 9 x 10 -9 M or less, about 5 x 10 -9 M or less, about 4 x 10 -9 M or less, about 3 x 10 -9 or less, about 2 × 10 -9 M or less, or about 1 x 10 -9 In certain non-limiting embodiments, K d is about 3 x 10 -9 M or less. In certain non-limiting embodiments, K d is about 1 x 10 -9 M ~ approx. 3×10 -7 In certain non-limiting embodiments, K d is about 1.5 x 10 -9 M ~ approx. 3×10 -7In certain non-limiting embodiments, K d is about 1.5 x 10 -9 M ~ approx. 2.7×10 -7 It's M.

[0133] The binding of the extracellular antigen-binding domain (e.g., in scFv or its analog) 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 protein-antibody complex of interest by using a reagent (e.g., antibody or scFv) specifically labeled for the complex of interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioisotopes can be detected by such means as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR 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).

[0134] In accordance with the subject matter of the present disclosure, a CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, where the extracellular antigen-binding domain specifically binds to an antigen, which may be a tumor antigen or a pathogen antigen.

[0135] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to CD 19. In certain embodiments, the CAR is one described in Kochenderder, JN et al. Blood. 2010 Nov 11;116(19):3875-86, the entirety of which is incorporated by reference. 3.3.1. Extracellular Antigen-Binding Domain of CAR

[0136] In certain embodiments, the extracellular antigen binding domain specifically binds to an antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is CD19. 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 a Fab, which is optionally cross-linked. In certain embodiments, the extracellular antigen binding domain is a F(ab)2. In certain embodiments, any of the foregoing molecules may be included in a fusion protein with a heterologous sequence 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. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is a pathogen antigen. 3.3.2.Transmembrane domain of CAR

[0137] 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. In accordance with the subject matter of the present disclosure, the transmembrane domain of the CAR can comprise a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.

[0138] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence or a fragment thereof 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: 17) (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, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 17 that is at least 20 amino acids long, or at least 30 amino acids long, or at least 40 amino acids long, or at least 50 amino acids long, and up to 235 amino acids long. Alternatively, or in addition, in various non-limiting embodiments, the CD8 polypeptide comprises or has an amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 137-209, 150-200, or 200-235 of SEQ ID NO: 17. In certain embodiments, the CAR comprises a transmembrane domain of CD8 (e.g., human CD8), or a portion thereof. In certain embodiments, a CAR of the present disclosure comprises a transmembrane domain that comprises a CD8 polypeptide that comprises or has an amino acid sequence of amino acids 137-209 of SEQ ID NO: 17. SEQ ID NO: 17 is provided below.

[0139] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV [SEQ ID NO: 17]

[0140] In certain embodiments, a CD8 polypeptide comprises or has an amino acid sequence or a fragment thereof 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: 18) (as used herein, homology may 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 has an amino acid sequence that is a contiguous portion of SEQ ID NO: 18 that is at least about 20 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, or at least about 60 amino acids in length, or at least about 70 amino acids in length, or at least about 100 amino acids in length, or at least about 200 amino acids in length, and up to 247 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD8 polypeptide comprises or has 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: 18. In certain embodiments, the CAR comprises the transmembrane domain of CD8 (e.g., murine CD8), or a portion thereof. In certain embodiments, a CAR of the present disclosure comprises a transmembrane domain that comprises a CD8 polypeptide that comprises or has the amino acid sequence of amino acids 151-219 of SEQ ID NO: 18. SEQ ID NO: 18 is provided below. [ka]

[0141] In accordance with the subject matter of this disclosure, a "CD8 nucleic acid molecule" refers to a polynucleotide that encodes a CD8 polypeptide.

[0142] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence or a fragment thereof 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_006130 (SEQ ID NO: 19), and / or can 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 has an amino acid sequence that is a contiguous portion of SEQ ID NO: 19 that is at least 20 amino acids long, or at least 30 amino acids long, or at least 40 amino acids long, or at least 50 amino acids long, and up to 220 amino acids long. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 153-179, or 200-220 of SEQ ID NO: 19. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 114-220 of SEQ ID NO: 19. In certain embodiments, the CAR comprises the transmembrane domain of CD28 (e.g., human CD28), or a portion thereof. In certain embodiments, the CAR comprises a CD28 polypeptide comprising or having amino acids 153-179 of SEQ ID NO: 19. SEQ ID NO: 19 is provided below. [ka]

[0143] An exemplary nucleic acid sequence encoding amino acids 153-179 of SEQ ID NO:19 is set forth in SEQ ID NO:20, provided below. TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG [SEQ ID NO: 20]

[0144] In certain embodiments, the transmembrane domain of the CAR of the present disclosure comprises a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence or a fragment thereof 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_031668.3 (SEQ ID NO: 21), and / or can 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 has an amino acid sequence that is a contiguous portion of SEQ ID NO: 21 that is at least 20 amino acids long, or at least 30 amino acids long, or at least 40 amino acids long, or at least 50 amino acids long, and up to 218 amino acids long. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-220, 150-200, 151-177, or 200-220 of SEQ ID NO:21. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 114-220 of SEQ ID NO:21. In certain embodiments, the CAR comprises the transmembrane domain of CD28 (e.g., murine CD28), or a portion thereof. In certain embodiments, the CAR comprises a CD28 polypeptide comprising or having amino acids 151-177 of SEQ ID NO:21. SEQ ID NO:21 is provided below. [ka]

[0145] In accordance with the subject matter of this disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide that encodes a CD28 polypeptide.

[0146] In certain non-limiting embodiments, the CAR can also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. The spacer region can be a hinge region from IgG1, or a CH2CH3 region of an immunoglobulin, and a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 19 or SEQ ID NO: 21), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 17 or a portion of SEQ ID NO: 18), a variant thereof that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to any of the foregoing, or a synthetic spacer sequence. 3.3.3.Intracellular signaling domain of CAR

[0147] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that can activate or stimulate a cell (e.g., a cell of lymphoid lineage, e.g., a T cell). Wild-type ("native") CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs) and transmits an activation signal to a cell (e.g., a cell of lymphoid lineage, e.g., a T cell) after antigen is bound. The intracellular signaling domain of the native CD3ζ chain is the primary transmitter of signals from the endogenous TCR.

[0148] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3ζ polypeptide. In certain embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence or a fragment thereof 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: 22), 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 CD3ζ polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 22 that is at least 20 amino acids long, or at least 30 amino acids long, or at least 40 amino acids long, or at least 50 amino acids long, and up to 164 amino acids long. Alternatively, or in addition, in various non-limiting embodiments, the CD3 ζ polypeptide comprises or has an amino acid sequence of amino acids 1-164, 1-50, 50-100, 100-150, 52-164, or 150-164 of SEQ ID NO: 22. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3 ζ polypeptide having amino acids 52-164 of SEQ ID NO: 22. SEQ ID NO: 22 is provided below. [ka]

[0149] In certain embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence or a fragment thereof 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_001106864.2 (SEQ ID NO: 23), 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 CD3ζ polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 23 that is at least about 20 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, or at least about 90 amino acids in length, or at least about 100 amino acids in length, and up to 188 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-142, 100-150, or 150-188 of SEQ ID NO: 23. SEQ ID NO: 23 is provided below. [ka]

[0150] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that comprises or has the amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NO: 24 is provided below. RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 24]

[0151] In certain embodiments, the intracellular signaling domain of the CAR comprises a mouse CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a human CD3ζ polypeptide.

[0152] 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.

[0153] In certain non-limiting embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling region. In certain embodiments, the costimulatory region comprises at least one costimulatory molecule that can provide optimal lymphocyte activation. As used herein, "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or their ligand that is required for a highly efficient response of lymphocytes to antigens. The at least one costimulatory signaling region can comprise a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. A costimulatory molecule can bind to a costimulatory ligand, a protein expressed on the cell surface, which upon binding to its receptor, generates a costimulatory response, i.e., an intracellular response that results in a stimulation that is provided when an antigen binds to the CAR molecule. Costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, and 4-1BBL. As an example, 4-1BB ligand (i.e., 4-1BBL) in combination with a CAR signal activates the CAR. + It can bind to 4-1BB (also known as "CD137") to provide an intracellular signal that induces effector cell function of T cells. CARs that contain intracellular signaling domains that include costimulatory signaling regions that include 4-1BB, ICOS, or DAP-10 are disclosed in U.S. Patent No. 7,446,190, the entirety of which is incorporated herein by reference.

[0154] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of CD28 or a portion thereof). In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a portion thereof). In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence, or a fragment thereof, 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 amino acid sequence set forth in SEQ ID NO: 19, and / or optionally comprises 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 has an amino acid sequence that is a contiguous portion of SEQ ID NO: 19 that is at least 20 amino acids in length, or at least 30 amino acids in length, or at least 40 amino acids in length, or at least 50 amino acids in length, and up to 220 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 181-220, or 200-220 of SEQ ID NO: 19. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 181-220 of SEQ ID NO: 19.

[0155] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of mouse CD28 or a portion thereof). In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence or a fragment thereof 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 amino acid sequence set forth in SEQ ID NO: 21, and / or optionally contains 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 has an amino acid sequence that is a contiguous portion of SEQ ID NO: 21 that is at least about 20 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, and up to 218 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-218, 115-218, 150-200, 178-218, or 200-218 of SEQ ID NO:21. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 115-218 of SEQ ID NO:21.

[0156] In accordance with the subject matter of this disclosure, a "CD28 nucleic acid molecule" refers to a polynucleotide that encodes a CD28 polypeptide.

[0157] In certain embodiments, the intracellular signaling domain of the CAR comprises the mouse intracellular signaling domain of CD28. In certain embodiments, the intracellular signaling domain of the CAR comprises the human intracellular signaling domain of CD28.

[0158] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises two costimulatory molecules: CD28 and 4-1BB or CD28 and OX40.

[0159] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human 4-1BB or a portion thereof. 4-1BB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. In certain embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence or a fragment thereof 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_001552 (SEQ ID NO: 25), and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO:25 that is at least about 20 amino acids long, or at least about 30 amino acids long, or at least about 40 amino acids long, or at least about 50 amino acids long, and up to 255 amino acids long. Alternatively, or in addition, in various non-limiting embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, 214-255, or 200-255 of SEQ ID NO:25. In certain embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence of amino acids 214-255 of SEQ ID NO:24. SEQ ID NO:25 is provided below. [ka]

[0160] In accordance with the subject matter of this disclosure, a "4-1BB nucleic acid molecule" refers to a polynucleotide that encodes a 4-1BB polypeptide.

[0161] In certain embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling domain of human 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling domain of mouse 4-1BB or a portion thereof.

[0162] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising an OX40 polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of OX40 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human OX40 or a portion thereof. In certain embodiments, the OX40 polypeptide comprises or has an amino acid sequence or a fragment thereof 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_003318 (SEQ ID NO: 26), and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the OX40 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 26 that is at least about 20 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, and up to 277 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the 4-1BB polypeptide comprises or has an amino acid sequence of amino acids 1-277, 1-50, 50-100, 100-150, 150-200, or 200-277 of SEQ ID NO: 26. SEQ ID NO: 26 is provided below. [ka]

[0163] In accordance with the subject matter of this disclosure, an "OX40 nucleic acid molecule" refers to a polynucleotide that encodes an OX40 polypeptide.

[0164] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising an ICOS polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of ICOS or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human ICOS or a portion thereof. In certain embodiments, the ICOS polypeptide comprises or has an amino acid sequence or a fragment thereof 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_036224 (SEQ ID NO: 27), and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, an ICOS polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 27 that is at least about 20 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, and up to 199 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, an ICOS polypeptide comprises or has an amino acid sequence of amino acids 1-277, 1-50, 50-100, 100-150, or 150-199 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below. [ka]

[0165] In accordance with the subject matter of this disclosure, an "ICOS nucleic acid molecule" refers to a polynucleotide that encodes an ICOS polypeptide. 3.3.4. Example CAR

[0166] In certain embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain that binds a CD19 polypeptide (e.g., a human CD19 polypeptide), a transmembrane domain that comprises a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof), an intracellular signaling domain that comprises a CD3ζ polypeptide, and a costimulatory signaling domain that comprises a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a portion thereof). In certain embodiments, a CAR is represented as "CD1928ζ". In certain embodiments, a CAR (e.g., CD1928ζ) comprises the amino acid sequence set forth in SEQ ID NO:28. SEQ ID NO:28 is provided below.

[0167] ALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVD FYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYN RYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 28]

[0168] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 28 is shown in SEQ ID NO: 29. SEQ ID NO: 29 is provided below.

[0169] 4.Cells

[0170] The subject matter of the present disclosure provides a cell comprising a dominant-negative Fas polypeptide disclosed herein. In certain embodiments, the cell further comprises an antigen-recognition receptor (e.g., CAR or TCR) that binds to an antigen. In certain embodiments, the dominant-negative Fas polypeptide is an exogenous dominant-negative Fas polypeptide. In certain embodiments, the antigen-recognition receptor can activate the cell. In certain embodiments, the dominant-negative Fas polypeptide (e.g., an exogenous dominant-negative Fas polypeptide) can promote the anti-tumor effect of the cell. The cell can be transduced with an antigen-recognition receptor and an exogenous dominant-negative Fas polypeptide, such that the cell co-expresses the antigen-recognition receptor and the exogenous dominant-negative Fas polypeptide.

[0171] In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a lymphocyte lineage cell. The lymphocyte lineage cell can provide antibody production, regulation of the cellular immune system, detection of foreign pathogenic agents in blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphocyte lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphocyte cells can be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0172] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the subject matter of the present disclosure 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 EMRAThe T cells may be of any type, including, but not limited to, 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 γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells may be genetically modified to target specific antigens by introduction of antigen recognition receptors, e.g., CARs or TCRs. In certain embodiments, the 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.

[0173] In certain embodiments, the cell is a virus-specific T cell. In certain embodiments, the virus-specific T cell comprises an endogenous TCR that recognizes a virus antigen. In certain embodiments, the cell is a tumor-specific T cell. In certain embodiments, the tumor-specific T cell comprises an endogenous TCR that recognizes a tumor antigen.

[0174] In certain embodiments, the cell is a NK cell. Natural killer (NK) cells are part of cell-mediated immunity and can be lymphocytes that act during natural immune response. NK cells do not require prior activation to carry out their cytotoxic effect on target cells.

[0175] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, e.g., Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express T cell receptor complexes that recognize full-length tumor antigens, including α and β heterodimers), Panelli, MC, et al. 2000 J Immunol 164:495-504; Panelli, MC, et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res. 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood 102:2498-2505 (disclosing the selectivity of in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPC) or pulsed dendritic cells). Immunoreactive cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0176] In certain embodiments, the cell is a myeloid lineage cell.Non-limiting examples of myeloid lineage cells include monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, megakaryocytes, platelets, and stem cells that can differentiate into myeloid lineage cells.In certain embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell or induced pluripotent stem cell).

[0177] The cells of the present disclosure can modulate the tumor microenvironment. Tumors have a microenvironment hostile to the host immune response, including a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes the infiltration-regulatory CD4+ These include 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 that target immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). These mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inappropriate immune responses, however, within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. Taken together, these immunosuppressive factors can induce either significant anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with targeted tumor cells.

[0178] In certain embodiments, the cells of the present disclosure have increased cell persistence, hi certain embodiments, the cells of the present disclosure have decreased apoptosis and / or anergy. 5. Compositions and Vectors

[0179] The presently disclosed subject matter provides compositions comprising a dominant negative Fas polypeptide disclosed herein (e.g., disclosed in Section 2) and an antigen recognition receptor disclosed herein (e.g., disclosed in Section 3). Also provided are cells (e.g., immunoresponsive cells) comprising such compositions.

[0180] In certain embodiments, the dominant negative Fas polypeptide is operably linked to a first promoter. In certain embodiments, the antigen recognition receptor is operably linked to a second promoter.

[0181] Furthermore, the presently disclosed subject matter provides nucleic acid compositions comprising a first polynucleotide encoding a dominant negative Fas polypeptide disclosed herein (e.g., as disclosed in Section 2) and a second polynucleotide encoding an antigen recognition receptor disclosed herein (e.g., as disclosed in Section 3). Cells comprising such nucleic acid compositions are also provided.

[0182] In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the dominant negative Fas polypeptide, hi certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the antigen recognition receptor.

[0183] 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, long terminal repeat (LTR) 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, IL-2 promoter, IL-12 promoter, p40 promoter and Bcl-xL promoter.

[0184] The compositions and nucleic acid compositions can be administered to a subject or / and delivered to cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells) can be achieved by transducing a substantially homogenous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (either a gamma retroviral vector or a lentiviral vector) is used to introduce the DNA construct into cells. For example, a first polynucleotide encoding an antigen-recognizing receptor and a second polynucleotide encoding a dominant-negative Fas polypeptide can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific to the target cell type of interest. Non-viral vectors can be used as well.

[0185] For the initial genetic modification of cells containing a dominant-negative Fas polypeptide and an antigen-recognizing receptor (e.g., CAR or TCR), a retroviral vector is generally used for transduction, however, any other suitable viral vector or non-viral delivery system can be used. The antigen-recognizing receptor and the dominant-negative Fas polypeptide can be constructed in a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, 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, Pestivirus 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). Also suitable are combinations of retroviral vectors and appropriate packaging lines, where the capsid protein is functional to infect 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. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles, such as those pseudotyped with VSVG, RD114 or GALV envelopes, and any others known in the art, are also suitable.

[0186] Possible methods of transduction include direct co-culture of cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culture with viral supernatant alone or with concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0187] Other transduction virus vectors can be used to modify cells.In certain embodiments, the vector selected shows high efficiency infection, and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, 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., 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., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).Retroviral vectors have been particularly well developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0188] Non-viral approaches can also be used for the genetic modification of cells. For example, nucleic acid molecules can be delivered by administration of nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987;Ono et al., Neuroscience Letters 17:259, 1990;Brigham et al., Am. J. Med. Sci. 298:278, 1989;Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988;Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990) into immunoresponsive cells. Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes can also be potentially useful for delivery of DNA into cells. Transplantation of normal genes into diseased tissues of subjects can also be achieved by introducing normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then the cells (or their progeny) are injected into targeted tissues or injected systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases or TALE nucleases, CRISPR). Transient expression may be obtained by RNA electroporation.

[0189] Any targeted genome editing method can also be used to deliver the dominant negative Fas polypeptide and / or antigen recognition receptor disclosed herein to cells or subjects.In certain embodiments, CRISPR system is used to deliver the dominant negative Fas polypeptide and / or antigen recognition receptor disclosed herein.In certain embodiments, zinc finger nuclease is used to deliver the dominant negative Fas polypeptide and / or antigen recognition receptor disclosed herein.In certain embodiments, TALEN system is used to deliver the dominant negative Fas polypeptide and / or antigen recognition receptor disclosed herein.

[0190] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, containing the RNA used by Cas9 to guide it to the correct part of the host DNA together with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and optional portions of DNA repair template (DNA that guides the cellular repair process allowing the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid that transfects the target cell. The crRNA is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell and therefore needs to be designed for each application. The repair template with 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 code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA), which can be spliced ​​together with the Cas9 gene into a plasmid for transfection into cells.

[0191] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that arise 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 that allow the zinc finger nuclease to target desired sequences in 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 way to generate new zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type II restriction endonuclease FokI. Using endogenous homologous recombination (HR) machinery and a homologous DNA template bearing the CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. When 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 cut ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0192] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems operate on almost the same principle as ZFNs. They arise by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a repeating motif of 33-34 amino acids 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 directing the nuclease to cleave at a specific position in the genomic DNA sequence.

[0193] Polynucleotide therapeutic methods can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), Simian Virus 40 (SV40) or metallothionein promoter) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, expression of the nucleic acid can be directed using enhancers known to preferentially direct gene expression in specific cell types. Enhancers used can include, but are not limited to, those characterized as tissue or cell specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequence or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0194] The method for delivering genome editing agent / system can be varied according to 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 via 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, envelope protein pseudotyping of viral vectors, cis- and trans-acting elements of replicable vectors, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles and cell-penetrating peptides).

[0195] The resulting cells can be grown under conditions similar to those for the unmodified cells, thereby allowing the modified cells to be expanded and used for a variety of purposes. 6. Polypeptides and analogues

[0196] The subject matter of the present disclosure also includes CD19, CD28, 4-1BB, CD8, CD3zeta and Fas polypeptides or fragments thereof that are modified in a manner that enhances their antineoplastic activity when expressed in immunoresponsive cells. The subject matter of the present disclosure provides a method for optimizing amino acid or nucleic acid sequences by generating changes in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure further includes analogs of any naturally occurring polypeptides disclosed herein, including but not limited to CD19, CD8, 4-1BB, CD28, CD3zeta and Fas. Analogs may differ from the naturally occurring polypeptides disclosed herein by amino acid sequence differences, 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 or identity with all or a portion of the naturally occurring amino acid sequence of the subject of the present disclosure. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, for example at least 25, 50 or 75 amino acid residues, or more than 100 amino acid residues. Again, in an exemplary approach to determine the degree of identity, a BLAST program is used to select sequences that show closely related sequences, e.g., -3 ~e -100A probability score between the . Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications can occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogs can also differ from naturally occurring polypeptides by changes in primary sequence. These include genetic variants, both natural and induced (resulting from random mutagenesis, for example, by exposure to irradiation or ethane methyl sulfate, or by site-directed mutagenesis, see Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids, or non-naturally occurring or synthetic amino acids, such as β or γ amino acids.

[0197] In addition to full-length polypeptides, the subject matter of the present disclosure also provides fragments of any one of the polypeptides or peptide domains disclosed herein. As used herein, the term "fragment" means 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 can be generated by methods known to those of skill in the art, or may be derived 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).

[0198] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins (e.g., dominant negative Fas polypeptides) 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 so that the resulting analog increases the antineoplastic activity of the original polypeptide when expressed in an immunoresponsive cell. These chemical modifications include, but are not limited to, substituting alternative R groups and changing the degree of saturation of certain carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to degradation in vivo and produce a longer therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below. 7. Administration

[0199] The cells of the present disclosure or compositions comprising the same can be provided to a subject systemically or directly to induce and / or enhance immune response to an antigen and / or to treat and / or prevent neoplasia and / or pathogen infection. In certain embodiments, the cells of the present disclosure or compositions comprising the same are directly injected into an organ of interest (e.g., an organ affected by neoplasia). Alternatively, the cells of the present disclosure or compositions comprising the same are provided indirectly to an organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase in vitro or in vivo generation of T cells or NK cells.

[0200] The cells of the present disclosure can be administered in any physiologically acceptable medium, usually intravascularly, although the cells may also be introduced into bone or other convenient sites (e.g., the thymus) where the cells may find a suitable site for regeneration and differentiation. Typically, at least about 1×10 5The final number of cells administered was approximately 1 × 10 10 or more. The cells of the present disclosure can include a population of purified cells. One of skill in the art can easily determine the percentage of the immunoresponsive 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 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%. The dosage can be easily adjusted by one of skill in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc.

[0201] The composition of the present disclosure may be a pharmaceutical composition comprising the cells of the present disclosure or their progenitors and a pharma- ceutically acceptable carrier. Administration may be autologous or heterologous. For example, cells or progenitors may be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived cells or their progeny (e.g., derived in vivo, ex vivo or in vitro) may be administered via catheter administration, systemic injection, local injection, intravenous injection or local injection, including parenteral administration. When administering the therapeutic composition of the present disclosure, it may be formulated into a unit dosage injection form (solution, suspension, emulsion). 8. Preparation

[0202] The composition comprising the cells of the present disclosure can be conveniently provided as a sterile liquid preparation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with specific tissues. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium, for example, containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0203] Sterile injectable solutions can be prepared by incorporating the genetically modified immunoresponsive cells in the required amount of an appropriate solvent with various amounts of other ingredients, if desired. Such compositions may be mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The compositions can also be lyophilized. Depending on the desired route of administration and preparation, the compositions can contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc. Suitable preparations can be prepared without undue experimentation by reference to standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference.

[0204] Various additives can 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 agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any medium, diluent or additive used must be compatible with genetically modified immunoresponsive cells or their precursor cells.

[0205] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears.The desired isotonicity of the composition can be achieved using sodium chloride or other pharma- ceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride can be particularly for buffers containing sodium ions.

[0206] The viscosity of the composition can be maintained at a selected level by using a pharma- ceutically acceptable thickening agent, if desired. 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 drug 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 a particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or another liquid form, for example, a sustained release form or a liquid-filled form).

[0207] The amount of cells administered will vary depending on the subject being treated. In one embodiment, about 10 4 ~about 10 10 Between pieces, about 105 ~about 10 9 Between pieces, or about 10 6 ~about 10 8 Between about 1×10 cells of the present disclosure are administered to a human subject. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1×10 8 pieces, approximately 2×10 8 pieces, about 3 x 10 8 pieces, about 4×10 8 Pieces or about 5 x 10 8 The cells of the present disclosure are administered to human subjects.The exact determination of what is considered to be an effective dose may be based on individual factors for each subject, including the size, age, sex, weight and condition of the particular subject.Dosage amount can be easily ascertained by those skilled in the art from the present disclosure and knowledge in the art.

[0208] One skilled in the art can easily determine the amount of cells and optional additives, vehicles and / or carriers in the composition and administered in the method. Typically, any additives (in addition to the active cells and / or drugs) are present in an amount of 0.001 to 50% (by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, e.g., about 0.0001 to about 5% by weight, about 0.0001 to about 1% by weight, about 0.0001 to about 0.05% by weight, or about 0.001 to about 20% by weight, about 0.01 to about 10% by weight, or about 0.05 to about 5% by weight. For any composition administered to animals or humans, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in an appropriate animal model, e.g., rodents such as mice: the dosage of the composition, the concentration of components therein, and the timing of administering the composition that elicits an appropriate response. Such determinations do not require undue experimentation from the knowledge of one of ordinary skill in the art, this disclosure, and the documents cited herein, and the times for sequential administration can be ascertained without undue experimentation. 9. Treatment Method

[0209] The subject matter of the present disclosure provides a method 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 to treat and / or prevent neoplasia in a subject. The cells of the present disclosure and compositions comprising the same can be used to prolong the survival of a subject suffering from neoplasia. The cells of the present disclosure and compositions comprising the same can also be used to treat and / or prevent neoplasia in a subject. The cells of the present disclosure and compositions comprising the same can also be used to reduce tumor burden in a subject. 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 disease in a subject, for example, an immunocompromised human subject. Such methods include administering an effective amount of the cells of the present disclosure or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, which is the alleviation of an existing condition or the prevention of recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided by bolus or continuous perfusion.

[0210] For adoptive immunotherapy using antigen-specific T cells, typically about 10 6 ~10 11 (e.g., about 10 9 A cell dose in the range of 100-1500 cells is injected. Upon administration of the cells of the present disclosure to the host and subsequent differentiation, T cells that are specifically directed against a specific antigen are induced. The modified cells can be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, intramedullary, and directly into the thymus.

[0211] The subject matter of the present disclosure provides a method for treating and / or preventing neoplasms in a subject.In certain embodiments, the method comprises administering an effective amount of the cell of the present disclosure or a composition comprising the same to a subject having a neoplasm.

[0212] In certain embodiments, the neoplasia or tumor is a cancer with increased FASLG RNA expression compared to normal tissue of a matched organ.See Yamamoto et al., J Clin Invest. (2019);129(4):1551-1565, which is incorporated herein by reference.

[0213] Non-limiting examples of neoplasia include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, pharyngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable carcinomas include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatocellular carcinoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor. Further included are any known in the field of oncology, including, but not limited to, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's hypergammaglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous cell and adenocarcinoma of the cervix, epithelial carcinoma of the uterus and ovary, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and scattered) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma. In certain embodiments, the neoplasia is selected from the group consisting of hematological cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, and pharyngeal cancer. In certain embodiments, the immunoresponsive cells of the present disclosure and compositions comprising same can be used to treat and / or prevent hematological cancer (e.g., leukemia, lymphoma, and myeloma) or ovarian cancer that is not amenable to conventional therapeutic intervention.

[0214] In certain embodiments, the neoplasm is a solid cancer or solid tumor. In certain embodiments, the solid tumor or solid tumor is selected from the group consisting of glioblastoma, prostate adenocarcinoma, renal papillary cell carcinoma, sarcoma, ovarian cancer, pancreatic adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, esophageal cancer, uterine endometrioid carcinoma, breast cancer, skin melanoma, lung adenocarcinoma, gastric adenocarcinoma, cervical cancer, renal clear cell carcinoma, testicular germ cell tumor and aggressive B-cell lymphoma.

[0215] The subject may have an advanced form of the disease, in which case the goal of treatment may include alleviating 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 therapy typically includes reducing or delaying the risk of recurrence.

[0216] Suitable human subjects for treatment typically include two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are subjects that carry 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 on their own are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to induce anti-tumor responses with the aim of alleviating the condition of the subject. Ideally, a reduction in tumor mass results, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of tumor progression, or a reduction in the pathological consequences of tumors.

[0217] The second group of suitable subjects is known in the art as the "adjuvant group". These are individuals who have a history of neoplasia but are responsive to other forms of treatment. 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 to be at risk for disease progression, either near the original tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on characteristics observed before and after initial treatment. These characteristics are known in the clinical field and are appropriately defined for each different neoplasia. Typical characteristics of the high-risk subgroup are those in which the tumor invades adjacent tissues or shows lymph node involvement.

[0218] Another group has a genetic predisposition to neoplasia, but does not yet have documented clinical signs of 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 herein in a prophylactic treatment to prevent the onset of neoplasia until it is appropriate to perform prophylactic surgery.

[0219] As a result of surface expression of an antigen-recognizing receptor that binds to tumor antigens, and a dominant-negative Fas polypeptide (e.g., an exogenous dominant-negative Fas polypeptide) that enhances the antitumor effect of cells that contain the antigen-recognizing receptor and a dominant-negative Fas polypeptide, adoptively transferred T cells or NK cells are endowed with enhanced selective cytolytic activity at the tumor site. Furthermore, after their localization to the tumor or viral infection and their proliferation, the T cells transform the tumor or viral infection site into a highly conductive environment for a wide range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) that are involved in the physiological antitumor or antiviral response.

[0220] In addition, the subject matter of the present disclosure provides a method for treating and / or preventing pathogen infection (e.g., viral infection, bacterial infection, fungal infection, parasitic infection or protozoal infection) in a subject, for example, in an immunocompromised subject. The method can include administering an effective amount of the cell of the present disclosure or a composition comprising the same to a subject with a pathogen infection. Exemplary viral infections that are susceptible to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) and influenza virus infection.

[0221] Further modifications can be introduced into the 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 when healthy tissue expresses the same target antigen as tumor cells, resulting in a similar outcome to GvHD. A potential solution to this problem is to engineer a suicide gene into the cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can allow T cell ablation by administering anti-EGFR monoclonal antibodies (e.g., cetuximab). The EGFRt can be covalently joined upstream of the antigen recognition receptor. The suicide gene can be included in a vector that includes a nucleic acid encoding the CAR of the present disclosure. In this method, administration of a prodrug (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9) designed to activate a suicide gene during T cell transformation of a malignant tumor (e.g., GVHD) causes apoptosis in suicide gene-activating receptor-expressing (e.g., CAR-expressing) T cells. Incorporation of a suicide gene into an antigen recognition receptor (e.g., CAR) of the present disclosure confers an additional level of safety by virtue of its ability to eliminate the majority of receptor-expressing (e.g., CAR-expressing) T cells in a very short period of time. Cells (e.g., T cells) of the present disclosure with an incorporated suicide gene can be preemptively eliminated at a given time point following T cell infusion or eradicated at the earliest sign of toxicity. 10. Kit

[0222] The subject matter of the present disclosure provides a kit for inducing and / or enhancing immune response and / or treating and / or preventing neoplasms or pathogen infections in a subject. In certain embodiments, the kit comprises an effective amount of the cells of the present disclosure or a pharmaceutical composition comprising the same. In certain embodiments, the kit comprises a sterile container, which may be in the form of a box, an ampoule, a bottle, a vial, a tube, a bag, a sachet, a blister pack, or other suitable container known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding an antigen recognition receptor (e.g., CAR or TCR) directed toward an antigen of interest, and an isolated nucleic acid molecule encoding an expressible form of a dominant negative Fas polypeptide, which may be included in the same or different vectors as needed.

[0223] Optionally, 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 a pathogen or an immune disorder. The instructions generally include information about the use of the composition for the treatment and / or prevention of neoplasia or pathogen infection. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for the treatment or prevention of neoplasia, pathogen infection or immune disorder, or symptoms thereof; cautions; 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 as a label applied to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. EXAMPLES

[0224] The practice of the present disclosure employs, 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 the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "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); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and, as such, may be considered in making and practicing the presently disclosed subject matter. Techniques that are particularly useful for certain embodiments are discussed in the sections that follow.

[0225] 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. Example 1 T cells engineered to overcome death signaling within the tumor microenvironment enhance adoptive cancer immunotherapy Introduction

[0226] Multiple variables can influence the success or failure of transferred T cells to mediate cancer regression (15). These may include the state of T cell differentiation (16) and local immunosuppressive factors present within the tumor-bearing host (17). Despite these complexities, one of the single most consistent correlates of responses observed in both hematological (2-5, 7) and solid tumors (10, 18, 19) has been the expansion and / or persistence of transferred T cells following infusion.

[0227] We hypothesized that disruption of factors that negatively regulate T cell proliferation and survival could represent a potentially ready-to-use pathway to enhance adoptive immunotherapy. Several clinical trials have tested whether cell-exogenous approaches could improve the persistence of adoptively transferred T cells, including the coadministration of immune checkpoint inhibitors (20, 21). However, these agents may not always efficiently enter the microenvironment of solid tumors (22) and may cause nonspecific immune activation resulting in systemic toxicity that does not contribute to efficacy (23). Thus, cell-intrinsic strategies were explored to enhance function exclusively within tumor-specific T cells, thereby including the risk of systemic toxicity and taking full advantage of the ability to reliably genetically engineer human T cells for clinical applications.

[0228] Using a cross-cancer analysis to identify candidate ligands that can limit the ability of T cells to expand and persist within tumor-bearing hosts, we discovered that the canonical apoptosis-inducing ligand FASLG is preferentially expressed in the majority of human tumor microenvironments. Furthermore, most therapeutic T cells used constitutively for adoptive immunotherapy were found to express Fas, the cognate receptor for FasL. Based on these findings, we developed a series of Fas dominant-negative receptors (DNRs) that function in both primary mouse and human T cells to prevent FasL-induced apoptosis. Adoptively transferred Fas DNR-engineered T cells exhibited enhanced T cell persistence and antitumor immunity without resulting in uncontrolled lymphoproliferation. Taken together, these results provide a potentially universal strategy to enhance the durability and survival of adoptively transferred T cells in a wide range of human malignancies following ACT. Methods and Materials

[0229] Human specimens: Peripheral blood mononuclear cells (PBMCs) were obtained from age- and sex-matched healthy donors or from melanoma and diffuse large B-cell lymphoma (DLBCL) patients enrolled in adoptive immunotherapy clinical protocols. All anonymous NIH blood bank donors and cancer patients providing PBMC samples were enrolled in clinical trials approved by the NIH Clinical Center and NCI Institutional Review Boards. Each patient signed an informed consent form and received a patient information form prior to participation.

[0230] Cancer Genome Atlas (TCGA) cross-cancer bioinformatics analysis: RNA sequencing (RNA-seq) data from 26 human cancers from the TCGA dataset and matched normal tissues from the GTEx dataset were collected and analyzed by UCSC Xena in the form of RNA-seq normalized by expectation maximization (RSEM) values. FASLG gene expression was analyzed as normalized RSEM counts, respectively. Statistics were corrected by Mann-Whitney U test. To identify genes positively correlated with FASLG expression, pre-ranked gene set enrichment was performed on all KEGG pathways in the mSigDB database. Pearson correlation was performed on the top 1000 genes positively correlated with FASLG expression averaged across 26 TCGA histologies.

[0231] Mice: Adult 6-12 week old male or female C57BL / 6 NCR (B6; Ly5.2 + ) was purchased from Charles River Laboratories at NCI Frederick. a Pepc b / BoyJ(Ly5.1 + ), B6.129S7-Rag1Lm / Mom / J(Rag), B6.MRL-Fas l p r / J(lpr), B6.Cg-Thy1 a / Cy Tg(TcraTcrb)8Rest / J(pmel-1(67)), MRL / MpJ (MRL-Mp) and MRL / MpJ-Faslpr / J (MRL-lpr) mice were purchased from Jackson Laboratory. Where indicated, pmel-1 mice were crossed onto Ly5.1, Rag, or Rag × lpr backgrounds. All mice were maintained under specific pathogen-free conditions. Animal experiments were approved by the Institutional Animal Care and Use Committees of the NIC and were performed in accordance with NIH guidelines.

[0232] Retroviral vectors and mouse and human CD8 + Transduction of T cells: Mouse and human Fas cDNA sequences were synthesized and cloned separately (Genscript) into MSGV retroviral plasmids, preceded by a T2A skip sequence and the selection marker Thy1.1. Transduction of mouse T cells was performed as previously described (68). Briefly, Platinum-E allotropic packaging cells (Cell BioLabs) were plated on BioCoat 10 cm dishes (Corning) overnight prior to transfection. The following day, MSGV-Thy1.1 (empty), MSGV-WT-mFas-Thy1.1 (mWT), MSGV-I246N-mFas-Thy1.1 (Fas I246N ) or MSGV-ΔDD-mFas-Thy1.1 (Fas ΔDD ), or 24 ug of retroviral plasmid DNA encoding MSGV-1D3-28Z (anti-CD19 CAR) ( 71 ) were mixed separately with 6 ug of pCL-Eco plasmid DNA in OptiMEM without antibiotics in 10% medium applied to Platinum-E cells for 7 h, together with 60 uL of Lipofectamine2000 (ThermoFisher).

[0233] Plasmids encoding human Fas mutant genes were subcloned into a murine leukemia virus based SFG retroviral vector as described by Maher et al., Nat Biotechnol (2002);20:70-75.

[0234] Media was changed after 7 hours, and viral supernatant was harvested from cells after 48 hours and centrifuged to remove debris. Retroviral supernatant was spun at 2000xg for 2 hours at 32°C in non-tissue culture treated 24-well plates coated overnight with 20ug / mL Retronectin (Takara Bio). 24 hour activated CD8α +T cells were added to the plate from which all but 100 uL of viral supernatant had been removed, spun at 1500 rpm for 5 min at 32° C., and then incubated overnight. Transduction was repeated twice the following day as described above. For transduction of human T cells, 293T cells (69) and RD114 were used instead of Platinum-E cells, and transfection and virus harvest proceeded as described for mouse virus production.

[0235] T cell culture and Fas death assay: Human PBMCs from healthy donors or patients were obtained by either leukapheresis or venipuncture and centrifuged on a Ficoll-Hypaque (Lonza) gradient to remove red blood cells and isolate lymphocytes. Cells were washed twice with PBS containing 1 mM EDTA, stained with a fixative cell viability dye (Thermo Fisher) in PBS, then washed twice with PBS supplemented with 2% FBS and 1 mM EDTA (FACS buffer). Intact human CD8α + T cells were isolated using a human CD8 isolation kit (Stem Cell Technologies). Mouse and human T cells, as well as E2a-PBX leukemia cells (72), were maintained in RPMI1640 (Gibco) with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin / streptomycin (100 U / mL and 100 ug / mL, respectively; Gibco), gentamicin (10 ug / mL), MEM non-essential amino acids (Gibco), sodium pyruvate (1 nM), GlutaMAX (2 mM), 0.011 mM 2-mercaptoethanol, and amphotericin B (250 ng / mL). B16-mhgp100 tumor cells, platinum-E cells, and 293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS and the additives mentioned above.

[0236] Pristine mouse CD8α + T cells were analyzed using MACS CD8 +Spleen cells were isolated using a negative selection kit (Miltenyi Biotec) and stimulated with plate-bound anti-CD3 (2ug / mL, clone 145-2C11, BD Biosciences), soluble anti-CD28 (1ug / mL, clone 37-51, BD Biosciences) and IL-2 (5ng / mL) in tissue culture treated 24 well plates. pmel-1 T cells were stimulated with 1ug / mL human gp100(25-33) peptide and IL-2 (5ng / mL, Prometheus) in whole splenocyte cultures. Human PBMC or CD8α + T cells were stimulated with plate-bound anti-CD3 (1 ug / mL, clone OKT3, BD Biosciences), soluble anti-CD28 (1 ug / mL, clone CD28.2, BD Biosciences) for 2 days, then given IL-2 (20 ng / mL) for the remainder of the culture. Cells were stimulated for 24 h before transduction with viral supernatant on days 1 and 2 of culture. On day 3, cells were removed from retronectin-coated plates and returned to tissue culture-treated 24-well plates or flasks. Where mentioned, cells were grown with either vehicle or the indicated concentrations of lz-FasL, a recombinant form of oligomerized FasL (43, 52). Five to six days after stimulation, T cells were washed twice with PBS and plated at 1–2 × 10 in 24-well plates with the indicated concentrations of lz-FasL. 5 Cells were plated at 10 cells / well and incubated for 6 or 24 hours at 37° C. with 5% CO2. Cells were then washed twice and stained with either Annexin V and PI positive or live / dead fixative dyes (Thermo Fisher), as well as CD8α (clone 53-6.7, BD Biosciences) and Thy1.1 (clone HIS51, eBioscience).

[0237] Flow cytometry, intracellular cytokine staining and phosphoflow: Cells were stained with a fixative cell viability dye (Thermo Fisher) in PBS and then washed twice with PBS supplemented with 2% FBS and 1 mM EDTA (FACS buffer). Cells were stained with the following fluorochrome-conjugated antibodies: CD3 (UCHT1), CCR7 (3D12), CD45RA (HI100), CD45RO (UCHL1), CD28 (CD28.2), CD95 (DX2) (BD Biosciences); and CD27 (M-T271), CD62L (DREG-56), CD8α (SK1), CD4 (OKT4) (BioLegend).

[0238] Mouse T cells, BM and splenocytes were stained with a fixative live / dead dye followed by the following antibodies: CD3 (145-2C11), CD8α (53-6.7), Vβ13 (MR12-3), Ly5.1 (A20), Ly5.2 (104), CD62L (MEL-14), CD95 (Jo2), B220 (RA3-6B2) (BD Biosciences); CD44 (IM7), CD19 (6D5), CD93 (AA4.1) (BioLegend); Thy1.1 (HIS51, eBioscience). For anti-CD19 CAR detection (67), biotin-protein L (Genscript) was utilized.

[0239] For phosphoflow, cells were fixed and permeabilized using BD Phosflow reagent according to the manufacturer's protocol. After permeabilization, cells were stained with pAkt (S473) (D9E) and pS6 (S235 / 236) (D57.2.2E) from Cell Signaling. For intracellular cytokine staining, cells were stained with fixative live / dead dye in PBS, then stained for surface antibodies in FACS buffer, then fixed, permeabilized (BD Biosciences), and stained for IFNγ (XMG1.2, BD Biosciences) and IL-2 (JES6-5H4, BioLegend). For FasL staining, tumor cells were stained with vehicle (PBS) or mouse IFN-γ (100 ng ml -1, Bio-Legend) for 24 h and then stained with FasL (Kay-10) and H-2Db (KH95) (BD Biosciences). All flow cytometry data were acquired using a BD Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo v.9.9 software (TreeStar).

[0240] Sanger sequencing analysis: Thy1.1 enriched empty vector or Fas I246N Genomic DNA from the transduced cells was extracted using the AllPrep DNA / RNA Mini Kit (QIAGEN). The primers (IDT) were I246N The point mutation was located upstream of Fas and the reverse primer was designed to locate the Thy1.1 reporter. PCR amplification (Invitrogen) was followed by Sanger sequencing.

[0241] Adoptive cell transfer, T cell enumeration and tumor treatment: For analysis of in vivo persistence, 6-12 week old male or female B6 mice received 6 Gy total body irradiation. One day later, they were injected with 5 × 10 T cells transduced with a reporter construct containing Thy1.1. 5 Congenically marked pmel-1 T cells were injected via tail vein injection. Mice were sacrificed on the indicated days and splenocytes were analyzed for homeostatic expansion of pmel-1 T cells.

[0242] For tumor treatment experiments, 6- to 12-week-old male or female B6 mice were transfected with the chimeric human / mouse gp100 antigen KVPRNQDWL. (SEQ ID NO:30) (aa25-33) overexpressing 5 × 10 5 previously described B16 melanoma line (57), or 1 × 10 6 CD19 +Tumor-bearing mice were injected with E2a-PBX leukemia cells. On the indicated days, tumor-bearing mice received 6 Gy total body irradiation. Mice were left untreated as controls or received the indicated doses of congenic marked pmel-1 or anti-CD19 CAR-transduced T cells modified with a reporter construct containing Thy1.1 by tail vein injection. To analyze the persistence and leukemia burden of anti-CD19 CAR-transduced T cells, mice were sacrificed 14 days later and cellular analysis in splenocytes and BM was performed.

[0243] For experiments in MRL-Mp mice, 8-week-old female mice received 6 Gy of total body irradiation. One day later, the mice were also transduced with 3 × 10 6 Anti-CD19 CAR-transduced CD8α + T cells were injected into mice. Age-matched MRL-lpr female mice were left unmanipulated as ALPS positive controls. All transduced T cells were bead-enriched to >92% purity using anti-Thy1.1 magnetic microbeads (Miltenyi Biotec) immediately prior to injection. All treated mice received daily injections of 12 μg IL-2 ip for 3 days. All tumor measurements were performed in a blinded manner by an independent investigator.

[0244] Co-culture assay of T cells and tumor cells: After approximately 6 days of culture, pmel-1 T cells were washed twice with PBS and plated in 96-well round-bottom plates at 5 × 10 per well. 4 T cells were plated at 100x the normal concentration in T cell medium without IL-2. T cells were incubated with either alone or plate-bound anti-CD3 / CD28 (2 μg ml -1 ) with 1.5 × 10 per well in 1:3 E:T. 5 The cells were incubated either with 100 ng / mL of B16-mhgp100 cells or with 100 ng / mL of lz-FasL. Cells were co-cultured for 6 or 24 hours, then washed and stained for cell viability.

[0245] ELISA assay: Serum antinuclear and anti-dsDNA antibody analysis was performed on 1:5 diluted sera and ELISA was performed according to the manufacturer's instructions (Alpha Diagnostic International).

[0246] Histopathology: Lung tissues were fixed in 10% buffered formalin and stained with H&E. Histological sections were scored in a blinded manner by an interpreting pathologist. Scoring was as follows: 0, no specific findings; 1, mild infiltration; 2, minimal infiltration; 3, moderate infiltration; 4, severe infiltration.

[0247] Statistical analysis: The product of perpendicular tumor diameters was plotted as the mean ± SEM for each data point, tumor treatment graphs were compared by using the Wilcoxon rank sum test, and animal survival analysis was evaluated using the log-rank Mantel-Cox test. For all other experiments, data were compared using either unpaired two-tailed Student's t-test corrected for multiple comparisons with Bonferroni adjustment, or repeated measures using one-way or two-way ANOVA, as indicated. In all cases, a P value of less than 0.05 was considered significant. Statistics were calculated using Prism 7 GraphPad software (GraphPad Software Inc.). result The human tumor microenvironment overexpresses the death-inducing ligand FASLG

[0248] Across human ACT clinical trials for both hematological and solid cancers, in vivo T cell expansion and persistence positively correlated with clinical response (3-5, 10, 19). These observations led to the hypothesis that disruption of pathways that impair T cell proliferation and survival may represent potential actionable targets for improving prognosis after adoptive transfer. To determine whether ligands that negatively modulate T cell proliferation and survival are enriched within the human tumor microenvironment, RNA sequencing data were compared using tumor-containing samples from the TCGA database compared to matched normal tissue of origin. Given recent evidence that tissue adjacent to resected tumors has an inflamed transcriptome profile that reflects an intermediate state between transformed and nontransformed tissues (24), expression data from the Genotype-Tissue Expression (GTEx) database (25) were used as normal controls. In total, 9,330 samples from 26 different cancer types for which appropriately matched tissue of origin was available were analyzed (Table 1). Raw data from each dataset were extracted and normalized in an identical manner using RNA-Seq with the random-regression expectation maximization (RSEM) method ( 26 ).

[0249] [Table 1-1] [Table 1-2]

[0250] We found that expression of FASLG, the gene encoding the canonical inducer of cellular apoptosis FasL (CD178), was overexpressed in the majority of evaluated cancer types compared to normal tissues (Figure 1A). This included both immunotherapy-responsive cancers such as cutaneous melanoma (SKCM), renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), and gastroesophageal carcinoma (STAD / ESCA), as well as cancers relatively resistant to current immunotherapies such as breast cancer (BRCA), colorectal adenocarcinoma (READ / COAD), glioblastoma multiforme (GMB), ovarian cancer (OV), pancreatic adenocarcinoma (PAAD), and prostate adenocarcinoma (PRAD). In total, 73% (19 / 26) of the evaluated human tumor types showed significant differential expression of FASLG within the tumor volume compared to normal tissue controls (P<0.05–P<0.001; Mann-Whitney U test, Bonferroni correction). In contrast, only 19% (5 / 26) of cancer types showed no significant differential expression, and only a minority (8%; 2 / 26) showed evidence of reduced FASLG expression in tumor samples versus normal tissues.

[0251] To gain more insight into the nature of FASLG expression within the human tumor microenvironment, we performed a gene set enrichment analysis (GSEA) (27) using genes positively correlated with FASLG across all 26 evaluated cancer types (Figure 1B). Expression profiles for many tumor-associated pathways, including NK cell cytotoxicity, antigen processing and presentation, TCR signaling, primary immune deficiency, and apoptosis, were significantly enriched (normalized P value < 0.001, FDR q value < 0.001), respectively. Consistent with these findings, investigation of the top 200 genes positively correlated with FASLG revealed dominant markers related to both lymphocyte activation, such as IFNG, PRF1, 41BB, and ICOS, and immune counterregulation, such as PDCD1, LAG3, and IL10RA (Figure 1C and Table 2). Taken together, these data indicated that death-inducing ligands, which can impair T cell survival, are significantly overexpressed in the majority of human cancer microenvironments and highly correlated with expression signatures of immune activation and regulation.

[0252] [Table 2-1] [Table 2-2]

[0253] We next determined whether Fas (CD95), the cognate receptor for FasL, is expressed on the surface of T cells used for clinical adoptive immunotherapy. Fas was previously found to be expressed on all non-naive human T cell subsets from healthy donors (HDs), including central memory T cells (TCMs), effector memory T cells (TEMs), and effector memory T cells co-expressing CD45RA (TEMRA) (28, 29). CD8α + We analyzed the frequency of T cell subsets and Fas expression of each subset in patients with melanoma and aggressive B cell lymphoma from apheresis products used to generate therapeutic T cells for ACT. In these patients, we found high expression of Fas in TCM, TEM and TEMRA subsets (Figures 1D and 1E). In addition, naive CD8α expression in these patients versus age-matched HD groups was significantly higher than that in the control group. + We compared the frequency of Fas T cells (TN) in HD patients compared with melanoma and lymphoma patients. - It was found to have a significantly higher percentage of TN cells (Figure 1F), likely reflecting the influence of previous immune stimulation and lymphocyte depletion therapy in the analyzed cancer patients (5, 30, 31). Thus, a significant proportion of human T cells used for ACT express known death receptors, and these cells were transferred into a tumor microenvironment enriched for the expression of their cognate ligands. T cells engineered with a Fas dominant-negative receptor prevent FasL-mediated apoptosis

[0254] This finding indicated that patient-derived T cells used for adoptive immunotherapy were biased towards the Fas-expressing subset that was subsequently transferred into the FASLG-enriched tumor microenvironment. Based on these data, we next investigated whether disruption of Fas signaling in adoptively transferred T cells could prevent their apoptosis and improve their persistence in vivo. In addition to triggering T cell apoptosis, FasL is also an essential effector molecule for T cell-mediated tumor killing (32). Furthermore, systemic administration of either anti-FasL antibodies or Fas-Fc fusion proteins was associated with the development of lymphoproliferative syndrome and double-negative (DN) CD3 + B220 + CD4 - CD8 - TCR alpha / beta + This can induce toxicity, including the accumulation of abnormal populations of lymphocytes. (33, 34) For these reasons, cell-specific genetic engineering strategies were explored to disable Fas signaling only in tumor-reactive T cells, in order to maintain antitumor efficacy and minimize the risk of systemic toxicity.

[0255] Physiologically, FasL initiates apoptotic signaling by first inducing oligomerization of the Fas receptor into trimers or larger oligomers at the plasma membrane (Figure 2A) (35). Fas oligomers recruit the associated intracellular adaptor molecule Fas via the death domain (FADD) through a homologous death domain (DD) present in each molecule (36, 37). Aggregation of FADD recruits the cysteine-aspartic acid protease procaspase 8 (38) through a homologous death effector domain in each molecule to form the death-inducing signaling complex (DISC) that can initiate the apoptotic signaling cascade (39). Based on this mechanism of action, we hypothesized that overexpression of a mutant Fas variant genetically altered to prevent FADD binding would function as a dominant-negative receptor (DNR) when expressed in Fas-competent wild-type (WT) T cells used for adoptive immunotherapy. Currently, viral-based constructs are the most commonly used method to stably modify human T cells for clinical applications (40). Thus, a mouse Fas sequence in which any asparagine residue is substituted for isoleucine at position 246 of the DD (Fas I246N ), a naturally occurring mutant of mouse Fas that is unable to bind FADD ( 41 , 42 ), or a Fas mutant in which most of the intracellular DD has been truncated to prevent FADD binding (del aa222–306; Fas ΔDD A series of retroviral constructs were generated that encoded the Fas (Figures 2A and 7A). As controls, an empty vector construct and the complete WT sequence of Fas (Fas WT ) were generated. To identify transduced cells, all vectors contained a Thy1.1 reporter separated from Fas using the T2A "self-cleaving" sequence.

[0256] T cells were isolated from Fas-competent WT mice, activated in the presence of IL-2, and subjected to either empty, Fas WT , Fas I246N or Fas ΔDDPhenotypic analysis 6 days after activation and transduction revealed high transduction efficiency for all constructs as measured by Thy1.1 expression (Figures 7B and 7C). Notably, ectopic Fas expression reduced the expression of Fas cells by 5.1-fold higher than that of WT (6.8-fold higher Fas MFI) or mutant Fas variants (Fas I246N and Fas ΔDD The Fas MFI was measurably higher than the endogenous level of Fas expression for constructs containing either FasL or Fas-binding domains (43- and 98-fold higher Fas MFI for FasL, respectively) (Fig. 7B and 7D). After 6 days in culture, transduced T cells were stimulated with recombinant FasL molecules oligomerized through the leucine zipper domain (lz-FasL) to mimic the function of membrane-bound FasL (43) or left untreated as control. In the absence of lz-FasL, T cells transduced with each construct remained at similar viability (Fig. 2C). However, after exposure to lz-FasL, the viability of either the empty vector control or Fas WT Thy1.1 transduced with either + A significant proportion of T cells express apoptotic annexin V + PI + The Fas population was transformed into a splenic ... WT Overexpression of Fas consistently resulted in higher levels of apoptosis compared to empty vector-transduced T cells, indicating that expression of Fas above physiological levels sensitizes T cells to FasL-mediated cell death. I246N or Fas ΔDD T cells transduced with either of the vectors were almost completely protected from lz-FasL-induced apoptosis. I246N or Fas ΔDD Among pools of T cells transduced with Thy1.1, protection from apoptosis was observed + This is because the Fas DNR is restricted to the Fas subpopulation, indicating a cell-intrinsic function (Fig. 11). I246N and Fas ΔDDalso showed that FasL can protect neighboring T cells from apoptosis, possibly by functioning as a local FasL "sink." I246N No functional or genetic evidence of reversion to the WT sequence was found in T cells modified with Fas after successive in vitro restimulation. WT Compared with Fas I246N Selective enrichment for T cells modified with DNR was measured, showing that DNR remained functionally intact over time (Figures 12A and 12B). Furthermore, sequential restimulation of Fas I246N Sanger sequencing of transduced T cells showed no evidence of reversion of the I246N point mutation to the WT Fas sequence (Figures S12C and S12D). Thus, overexpression of Fas variants abolished their ability to bind FADD function in a dominant-negative manner that prevented FasL-mediated apoptosis in WT T cells.

[0257] Finally, we sought to determine whether Fas DNR offered protection from other apoptosis-inducing stimuli that adoptively transferred T cells may encounter in vivo. These include activation-induced cell death (AICD), cytokine withdrawal, and proximity to tumor cells. For these assays, we utilized pmel-1 T cells specific for the cancer antigen gp100, and B16 melanoma (B16 cells) engineered to express human gp100. Although B16 cells did not express FasL upon resting, FasL expression was measurably upregulated after incubation with IFN-γ (Figure 13). Fas I246N or Fas ΔDD pmel-1 T cells transduced with were equally protected from apoptosis induced by either lz-FasL or tumor coculture (Figure 14). In contrast, Fas ΔDD Transduction of T cells with Fas I246NThese findings suggest that Fas binds to FADD with reduced efficacy under certain conditions. I246N This disclosure therefore assumes that Fas is the only marker for all in vivo experiments that owes its superior functional attributes to the Fas ΔDD We focused solely on DNR, which allowed us to more clearly determine the impact of ablation of Fas signaling on the in vivo function of adoptively transferred T cells. Adoptive transfer of Fas DNR-engineered T cells results in superior persistence

[0258] It was next determined whether expression of Fas DNR in T cells resulted in superior in vivo persistence after adoptive transfer into tumor-bearing hosts.

[0259] Congenically marked, genetically modified pmel-1 T cells were cultured in sublethally irradiated Thy1.1 - The cells were adoptively transferred into C57BL / 6 (B6) mice to induce homeostatic proliferation, and the time-dependent expansion and persistence of the transferred cells were measured. ΔDD or empty vector control transduced T cells were identified by expression of the Thy1.1 reporter gene. To measure T cell proliferation, T cells were co-stained for the cell proliferation marker Ki-67.

[0260] One day after transfer, Fas ΔDD pmel-1 and empty vector modified T cells engrafted at similar levels and expressed Ki-67 nearly uniformly (Figures 3F-3H). Beginning within 3 days of transfer, multi-logarithmic expansion of both populations of modified cells was measured. However, at the peak of expansion, there was a significant increase in the expression of Fas ΔDDAn approximately 50-fold increase in the number of modified T cells was observed. This in turn led to a 10-fold higher level of persistence of Fas DNR modified T cells at day 30 (Figures 3F and 3G). Over time, a comparable reduction in Ki-67 expression in both engineered T cell populations was observed (Figure 3H), which correlated with reconstitution of the host's endogenous T cell compartment. These data suggested that in vivo proliferation was comparable between the two engineered T cell populations. However, Fas DNR modified T cells demonstrated superior overall expansion and mid-term persistence, likely due to reduced apoptosis.

[0261] We next sought to determine whether genetic modification with Fas DNR resulted in superior T cell persistence within the TME. Co-injection experiments were performed to ensure that modified T cells were exposed to the same microenvironmental factors within any given tumor.

[0262] Congenitally distinct pmel-1 CD8D specific for the cancer antigen gp100 + T cells, Ly5.1 - / Thy1.1 - or Ly5.1 + / Thy1.1 - The cells were obtained from either Fas ΔDD Transduced T cells expressing Thy1.1 were then purified using anti-Thy1.1 microbeads, recombined at an approximate 1:1 ratio, and then transduced with sublethally irradiated Ly5.1 bearing established B16 melanoma tumors for 10 days. - / Thy1.1 - The recipient mice were then co-injected with the adoptively transferred genetically modified Thy1.1 IL-2 antibody (Figure 3A). Treated mice received a limited course of IL-2 after transfer, as is currently done in many ACT clinical trials for solid tumors (13, 18, 44-46). Seven days after injection, both the spleens and tumors of the recipient mice were harvested and co-injected with the adoptively transferred genetically modified Thy1.1 IL-2 antibody (Figure 3B). +pmel-1 T cells were analyzed. Both in the spleens and tumors of recipient mice, Ly5.1 - Thy1.1 + Compared with empty vector modified T cells, Ly5.1 - Thy1.1 + Fas ΔDD We consistently found significant enrichment of engineered T cells (Figures 3B and 3E; P<0.01, P<0.001). ΔDD To test whether DNR-manipulated T cells could enhance T cell survival in a tumor cell-enriched microenvironment, an in vitro co-culture assay was performed. ΔDD pmel-1 T cells expressing either Thy1.1 or an empty vector control were plated overnight in the absence of IL-2 alone or co-cultured with B16 melanoma tumors. As a positive control for cell death, T cells were cultured in the presence of lz-FasL. In this experiment, T cells were not bead-enriched for Thy1.1 allowing for an additional internal control. After 24 hours, T cell viability was assessed by FACS analysis. Significant cell death was induced in empty vector-transduced pmel-1 T cells by either co-culture with B16 or addition of lz-FasL, which was consistent with the Fas ΔDD This was not observed in the transduced counterparts (Figure 3C). Also, non-transduced cells in both groups showed comparable cell viability in response to B16 coculture or lz-FasL (Figure 3D). Together, these results demonstrated that genetic manipulation with Fas DNR enhanced the engraftment and survival of tumor-reactive T cells after adoptive cell transfer and exposure to a tumor-rich microenvironment. ACT of Fas DNR-modified T cells does not result in an ALPS phenotype

[0263] Mice and humans with germline defects in components of normal apoptotic signaling, such as Fas, can develop profound alterations in normal lymphocyte homeostasis and development. These disorders, collectively referred to as autoimmune lymphoproliferative syndrome (ALPS), are characterized by abnormal CD3 + B220 + CD4 - CD8- These include the development of autoantibodies that result in accumulation and reduced survival of lymphocyte populations (47, 48). Given safety concerns that may be related to the abrogation of normal Fas signaling in mature T cells, Fas ΔDD Detailed long-term immune monitoring of animals that received DNR-modified T cells was performed (Figure 4E). This time point was chosen as mice with germline defects in Fas typically develop overt clinical symptoms within 3.5–5 months of age, depending on the background strain (49, 50). Unmanipulated WT and Fas-deficient lpr / lpr mice were used as negative and positive controls, respectively, for the ALPS phenotype, and Fas ΔDD Vβ3 13 modified with DNR or empty vector control + CD3 in the spleens of mice that had previously received ACT of pmel-1 T cells + B220 + Lymphocyte frequency was assessed. As expected, spleens from lpr / lpr mice showed abnormal CD3 + B220 + We found that mice receiving either the empty vector control or Fas DNR modified T cells showed a significant accumulation of lymphocytes (Figures 4A and 4B; P<0.05, P<0.001). In contrast, mice receiving either the empty vector control or Fas DNR modified T cells showed no significant increase in this population. To exclude transformation of our modified T cell population, we used the transferred Vβ3 13 + Thy1.1 + The long-term persistence and phenotype of engineered T cells were evaluated. ΔDD DNR-engineered T cells persisted in higher numbers than cells engineered with the empty vector control (Figures 4C and 4D; P<0.05). Long-lasting Fas DNR-engineered T cells express conventional CD3 + B220 - These data demonstrated that adoptively transferred pmel-1 T cells expressing Fas DNR did not undergo aberrant lymphoproliferation in B6 hosts.

[0264] It has previously been shown that expression of a transgenic TCR crossed with a Fas-deficient lpr background can limit the development of ALPS (74). In addition, the B6 strain develops lymphoproliferative symptoms at a slower rate compared to other strains (49, 50, 75). Thus, Fas ΔDD Additional experiments evaluating the safety of the DNR modification were performed by adoptively transferring open T cell repertoires engineered with either Fas DNR or a blank control into the ALPS-susceptible MRL-Mp strain. Fas-deficient mice on the MRL background (MRL-lpr mice) developed autoantibodies, nephritis, and splenomegaly in a more severe manner and several months earlier than B6-lpr mice (Figure S15A) (49,50,75). To induce activation and expansion of adoptively transferred T cells in this model, open-repertoire T cells from MRL-Mp mice were transfected with a previously described second-generation anti-CD19 28ζ CAR (71) and Fas ΔDD or control vector. The use of the anti-CD19 CAR in these experiments was consistent with the inhibition of host CD19 + promoted robust in vivo proliferation of T cells through B cell recognition. Of note, recently published data indicate that CAR-modified T cells can still be stimulated by their TCR ( 72 , 76 ).

[0265] Cells received either no (PBS) or anti-CD19 CAR transduced with FasΔDD or empty vector. + Spleens from MRL-Mp mice that received T cells were analyzed and compared to spleens from age-matched Fas-deficient MRL-lpr mice (FIG. 15C). Spleens from age-matched MRL-lpr mice were significantly heavier when compared to spleens from all other treatment groups. Importantly, PBS-treated mice, as well as Fas ΔDD No differences in spleen size were observed between mice receiving either anti-CD19 CAR-transduced cells modified with or without control. Flow cytometry analysis of splenocytes revealed unusual DN CD3 in the spleens of MLR-lpr mice, which together accounted for more than 30% of all lymphocytes.+ B220 + Strong lymphocyte expansion was demonstrated (Figures 15D and 15E). In contrast, empty vector and Fas ΔDD CD3 in T cell-treated mice + B220 + Lymphocyte frequencies were similar to levels observed in PBS control mice.

[0266] To assess the development of autoimmunity, serum analysis of all treated animals was performed using samples from MRL-lpr mice as a positive control. ΔDD or anti-CD19 CAR engineered with an empty vector + Mice that received T cells had low antinuclear and anti-dsDNA antibody titers comparable to PBS controls (Figure 15F). In contrast, sera from MRL-lpr positive control mice demonstrated high titers of both types of autoantibodies. Anti-CD19 CAR co-transduced with Fas DNR in the absence of uncontrolled lymphoproliferation and autoantibody formation. + T cells were treated with control modified anti-CD19 CAR + Compared to T cells, the CAR persisted at significantly higher levels in the spleens of recipient MRL-Mp mice (Figure 15G). Furthermore, persistent Fas DNR-modified CAR + T cells were cultured with a control modified CAR + Abnormal CD3 + B220 + These results suggest that Fas transferred to B6 hosts did not result in a higher percentage of cells receiving the IFN-γ antibody (Fig. 15H). ΔDD The findings were directly mirrored using modified pmel-1 T cells (Figures 4C and 4D).

[0267] Finally, blinded pathological evaluation of H&E-stained lung specimens was performed to assess whether ALPS-susceptible MRL-Mp recipient mice developed pulmonary pathology following adoptive transfer of Fas DNR-modified T cells. Consistent with previous reports (77), Fas-deficient MRL-lpr mice developed dense mononuclear inflammatory pulmonary infiltrates in perivascular and peribronchiolar regions (Figures 16A and 16B). In contrast, mice treated with FasΔDD or control-modified T cells did not display evidence of increased inflammatory infiltrates compared to PBS-treated control injections. Furthermore, no evidence of pulmonary fibrosis was observed.

[0268] Taken together, these data in both B6 and MRL-Mp strains support the notion that Fas ΔDD Despite increased relative survival of DNR T cells, evidence of unregulated lymphoid accumulation, Thy1.1 + CD3 + B220 + We demonstrated that no formation of Fas clusters or clinical evidence of autoimmunity was detected. Based on these data, infusion of mature T cells with impaired Fas signaling does not result in an acquired lymphoproliferative phenotype. T cell-intrinsic disruption of Fas signaling enhances antitumor efficacy following ACT

[0269] Having established that adoptively transferred T cells engineered with Fas DNR resulted in enhanced persistence without long-term toxicity, we next assessed the antitumor efficacy of these cells. pmel-1 T cells were stimulated and expressed Fas I246N , Fas ΔDDor an empty vector control. This was followed by restimulation and further expansion to mimic the more differentiated T cell populations present in the circulation of cancer patients (5, 31) (Figures 1D and 5A). Eleven days later, transduced T cells from each condition were isolated to >98% purity using anti-Thy1.1 microbeads and then injected separately into sublethally irradiated mice bearing established B16 melanoma tumors. Treated mice also received IL-2 by i.p. injection. Compared to untreated controls, all mice receiving adoptively transferred pmel-1 T cells experienced a significant delay in tumor growth (Figure 5B). However, Fas I246N or Fas ΔDD These mice that received T cells engineered with either DNR showed enhanced tumor control ( Fig. 5B ; P < 0.001) and significantly improved animal survival compared with control modified pmel-1 cells ( Fig. 5C ; P < 0.05 and P < 0.01).

[0270] It has recently been discovered that Fas stimulation can induce nonapoptotic Akt / mTOR signaling, leading to increased T cell differentiation (51, 52). Consistent with previous results, exposure to lz-FasL significantly increased phosphorylated (p)Akt in T cells transduced with the empty vector control. S473 and pS6S235,S236 It was found that IL-1 induced a dose-dependent increase in IL-1 (Figures 8A and 8B).

[0271] Expansion of control modified cells resulted in the accumulation of TEM-like cells with a reduced ability to produce IL-2 (Figures 8C and 8D). In contrast, FasI246N or Fas ΔDDT cells transduced with either Fas DNR or Thy1.1 did not show Akt and S6 phosphorylation and were protected from increased Akt-mediated T cell differentiation. These cells maintained a dominant TCM-like phenotype and the ability to produce IL-2. In several different animal models (29, 53, 54) and clinical trials (10, 55), transfer of TCM-like cells was associated with superior tumor regression compared with transfer of T cell-grade cells. These findings raised the possibility that the superior tumor regression observed with Fas DNR-modified cells might be due to differences in cell differentiation rather than protection from Fas-mediated T cell death. To test this possibility, the differentiation status of T cells was modified by transducing >96% pure cells of TCM-like phenotype (Thy1.1 + CD44 hi g h CD62L + ) by FACS sorting at the time of cell injection (Figure 5D). Central memory-like sorted T cells were then transferred into sublethally irradiated B16 tumor-bearing mice as described in Figure 5A. Even when normalized for TCM-like differentiation state, it was found that adoptive transfer of Fas DNR-modified T cells resulted in superior tumor regression and animal survival compared to control-modified T cells (Figures 5E-5H; P<0.05).

[0272] Taken together, prevention of Fas-mediated cell death in adoptively transferred, Fas DNR-engineered tumor-reactive T cells results in superior tumor regression and animal survival. Genetic manipulation with Fas DNR protects human T cells from Fas-mediated apoptosis

[0273] To determine the clinical viability of engineering human T cells with Fas DNR, we designed a retroviral construct encoding a human Fas sequence mutated to prevent FADD binding. This was a human Fas variant (hFas) containing a point mutation substituting a valine for the aspartic acid residue at position 244. D244V) ( 56 , 57 ), and human Fas with a large portion of its intracellular death domain truncated (del aa 230–314; hFasΔDD) ( Fig. 6A ) ( 56 , 57 ).

[0274] CD8 + T cells were isolated from HD PBMCs and stimulated with anti-CD3 / CD28 and IL-2, followed by hFas D244V , hFas ΔDD or an empty vector control (Figure 6B). - and transduced Thy1.1 + Both T cells remained similarly viable as measured by Annexin V and PI staining (Fig. 6C). However, when these cells were cultured in the presence of increasing doses of lz-FasL, empty vector-transduced T cells showed increased viability compared to the control T cells, as measured by Annexin V and PI staining. + A significant and dose-dependent increase in the frequency of apoptotic and necrotic cells was observed (Figures 6C and 6D). In contrast, hFas D244V or hFas ΔDD T cells modified with either lz-FasL or Thy1.1 were significantly protected from lz-FasL-mediated apoptosis. This protection was greater than that of non-transduced Thy1.1 - The cells were D244V or hFas ΔDD Transduced with Thy1.1 + A significantly higher frequency of annexin V compared to T cells + Thus, genetic engineering with Fas DNR protects primary human T cells from FasL-induced cell death and offers a new method to protect adoptively transferred T cells within the human tumor microenvironment. Consideration

[0275] The cross-cancer analysis results reported here strongly suggested that the canonical death-inducing ligand FASLG is overexpressed within the microenvironment of most human cancers. A significant proportion of human T cells used for adoptive immunotherapy co-expressed Fas, the cognate receptor for FasL. Based on these findings, we tested a cell-intrinsic strategy to "shield" Fas-competent mouse and human T cells from FasL-induced apoptosis using a series of Fas DNR-based genetic manipulations. Functionally adoptively transferred Fas DNR-modified T cells showed superior persistence in both the periphery and tumors of tumor-bearing animals, resulting in superior tumor regression and overall survival. Importantly, Fas DNR-modified T cells showed enhanced survival compared with control-modified T cells for up to 6 months after transfer, with no evidence of uncontrolled lymphoproliferation or autoimmunity detected. Thus, these findings provide a novel and potentially universal genetic manipulation strategy to enhance the function of adoptively transferred T cells against a broad range of human malignancies, including advanced solid tumors.

[0276] In addition to its canonical apoptosis-inducing function, it has previously been reported that Fas can also promote mouse and human T cell differentiation in an AKT-dependent manner (51, 52). Consistent with these findings, T cells transduced with Fas DNR express pAKT S473 and pS6 S235,S236 Thus, this blockade in AKT / mTOR signaling minimized T cell differentiation and promoted the accumulation of TCM-like cells that retained expression of the lymphocyte homing marker CD62L and the ability to produce IL-2. In multiple preclinical models (29, 53, 54) and in retrospective analyses of human clinical trials (10, 55), infusion of TCM-like cells was associated with superior antitumor outcomes compared with TEM-like cells. These findings raised the possibility that the superior treatment outcomes using Fas DNR-modified cells may have resulted from infusion of less differentiated T cells rather than prevention of apoptosis. To address this possibility, we performed a randomized controlled study of phenotypically matched FACS-sorted Fas DNR- or empty vector control-modified T cells. CMThe antitumor efficacy of Fas DNR-modified T cells was compared, even when normalized for surface phenotype. CM is the control modified T CM showed superior treatment efficacy compared to Fas DNR-modified T cells. Mechanistically, the primary cause of the enhanced in vivo antitumor efficacy using Fas DNR-modified T cells was due to disruption of cell death, but not due to the infusion of less differentiated cells. These findings are also consistent with recent reports from Zhu et al., Horton et al., and Lakins et al. (17, 58, 59) demonstrating FasL-induced apoptosis of tumor-infiltrating lymphocytes that limits the efficacy of immune checkpoint inhibitors.

[0277] Although analyses have shown that FASLG expression is enriched within the microenvironment of many human tumors, they do not define which specific cell types express the ligand. Using immunohistochemical protein staining, previous studies have identified that FasL can be expressed directly on the surface of many solid tumors identified in our cancer cross-sectional analysis. This includes breast, colon, brain, kidney, and cervical cancers (60, 61). In addition, recent studies have identified that FasL is expressed along the luminal surface of the peripheral cardiovascular system in human ovarian and brain cancers, creating a tumor endothelial death barrier that limits T cell infiltration (60, 62). Finally, it is possible that FasL can be expressed within the tumor microenvironment by cells of both the innate and adaptive immune systems. This possibility has been shown previously by others (17) and is further suggested by our own analyses demonstrating a high degree of correlation between FASLG and many immune-related genes. Finally, the functional data demonstrate that Fas DNR modification also provides protection from other apoptosis-inducing stimuli that T cells may experience following adoptive cell transfer into hosts bearing tumors or infections. These include activation-induced cell death (AICD), cytokine withdrawal, and proximity to antigen-expressing tumor cells. Collectively, these data suggest that the source of FasL may be dependent on tumor histology. Thus, a cell-specific Fas DNR approach that does not impair the FasL-mediated tumor killing capacity of transferred T cells may be broadly applicable to a variety of cancer types.

[0278] The Fas DNR now joins a list of other candidate DNRs that could be engineered to essentially disrupt signaling by immune suppressors present in the tumor microenvironment, including the TGFβ receptor (63) and PD1 (64). Disruption of Fas using a small hairpin RNA approach has been reported in human T cells in vitro (65), however, due to the relatively low efficacy of Fas knockdown, this approach required prolonged in vitro selection. Furthermore, the in vivo antitumor potential of these cells was not tested. Despite the enhanced cell persistence observed using Fas DNR-modified T cells, no evidence of double-negative T cell formation or uncontrolled lymphoproliferation was observed.

[0279] Germline loss of function in Fas signaling can result in autoimmune lymphoproliferative disease in both mice and humans, a potential safety consideration for the Fas DNR approach. Despite the increased survival of FasΔDD-modified T cells, we observed uncontrolled lymphoid accumulation, abnormal CD3 + B220 + No evidence of lymphoid cell population formation or autoimmunity was found, including by adoptive transfer of polyclonal T cell populations into the ALPS-prone MRL-Mp line. Based on these data, infusion of mature T cells with impaired Fas signaling is unlikely to result in an acquired lymphoproliferative syndrome.

[0280] Although Fas is a key mediator for initiating the extrinsic apoptosis signaling cascade, the intrinsic apoptosis pathway remains intact in the cell. Thus, competition for homeostatic cytokines, neglect due to the absence of antigen, and T cell exhaustion may all contribute to regulating the homeostasis of Fas DNR cells in vivo. Despite these reassuring safety data in mice, refinement of this approach for clinical application may include the introduction of a suicide mechanism, such as a truncated EGFR upstream of Fas DNR (66).

[0281] In conclusion, the FasL / Fas pathway is poised to become activated in many patients undergoing adoptive immunotherapy for the treatment of solid tumors. A novel dominant-negative receptor was developed that essentially abolishes the apoptosis-inducing function of this pathway in primary mouse and human T cells, resulting in enhanced cell persistence and increased antitumor efficacy. These data lay the groundwork for a potentially universal strategy to enhance the efficacy of adoptive immunotherapy for both solid and hematological tumors. Example 2 Effect of Fas DNR and anti-CD19 CAR-modified T cell treatment in a mouse model of leukemia

[0282] The therapeutic efficacy of adoptively transferred T cells engineered with both Fas DNR and CAR was next evaluated. An independent tumor model was used in which a hematological malignancy was targeted with CAR. A recently developed syngeneic B-cell ALL (B-ALL) line driven by a physiologically relevant E2a-PBX translocation was used in a treatment model using a second generation 28ζ anti-CD19 CAR in mice (72, 78). The syngeneic model was chosen over the more commonly used xenogeneic anti-CD19 CAR treatment model for two reasons. First, to ensure that the transferred T cells were fully responsive to host-derived FasL in addition to FasL expression by tumor cells and adoptively transferred T cells. Second, to avoid the potential confounding effect of xenogeneic reactivity on AICD induction in the transferred T cells.

[0283] T cells were stimulated and co-immunopreserved with anti-CD19 CAR and Fas ΔDD The co-transduction efficiency and purity of transduced T cells are shown in Figures 9B-9C and 10A-10B. Using protein L to identify CAR-transduced T cells (79), the co-transduction efficiency was determined by the expression of Fas ΔDDand empty vector control, followed by Thy1.1 bead enrichment, were similarly efficient. We next determined how co-transduced anti-CD19 CAR T cells responded to various apoptosis-inducing stimuli, including exogenous FasL, cytokine withdrawal, AICD, and exposure to antigen-expressing B-ALL tumor cells (Figure 10C). Similar to the results using TCR-expressing pmel-1 T cells, Fas ΔDD Expression of CAR transduced with empty vector control + Compared with T cells, CAR-modified T cells were protected from each of these death-inducing stimuli.

[0284] Anti-CD19 CAR and Fas in a mouse leukemia model ΔDD The experimental design for treatment with syngeneic T cells co-transduced with either CD4+ or empty vector control is shown in Figures 9A and 10D.

[0285] Treated mice received daily IL-2 injections for 3 days to support the expansion of adoptively transferred T cells. 14 days after cell injection, the two disease sites for E2a-PBX B-ALL, spleen and BM, were analyzed for the persistence of adoptively transferred cells. High levels of Thy1.1 were detected in both disease sites in comparison to mice that received empty vector-transduced T cells (Figure 10E). + Fas ΔDD E2a-PBX leukemias express classical pre-B-ALL markers, including CD19, B220, and CD93 (80). As shown in Figure 10F, the BM in untreated (PBS) and empty vector-treated mice contained approximately 70% leukemic cells 14 days after T cell treatment. However, Fas ΔDD Mice that received the modified cells contained less than 1% leukemia cells in the BM. These data support the conclusion that CAR + We showed that Fas DNR expressing T cells were able to mediate superior leukemia elimination compared with empty vector-transduced T cells.

[0286] After 11 days, transduced T cells from each condition were isolated to >98% purity using anti-Thy1.1 microbeads and then separately injected into sublethally irradiated mice bearing established E2a:PBX pre-B ALL tumors. Treated mice also received IL-2 by i.p. injection. Compared to untreated controls, high doses of CAR T cells (5.5×10 5 All mice receiving the low dose of CAR T cells (1.8 × 10) experienced a significant delay in tumor growth (Figure 9D). 5 When treated with ΔDD Only mice that received DNR-engineered T cells showed significantly improved animal survival compared to controls (Figure 9E).

[0287] In another experimental setting, two different doses of Fas ΔDD We analyzed survival of leukemia-bearing mice following adoptive transfer of second-generation 28ζ anti-CD19 CAR-transduced T cells co-modified with IL-1 or blank control. To provide a treatment window, we transferred a dose of CAR-modified T cells that was previously shown to be subtherapeutic in this model (72). Higher cell doses (3 × 10 5 pieces CAR + cells) and control or Fas ΔDD Modified CAR + Adoptive transfer of either T cell resulted in significantly improved animal survival compared to untreated mice (Figure 10G, left). However, the Fas DNR-modified CAR + All mice that received T cells survived, whereas control modified CAR + Mice that received CAR T cells did not survive longer than 55 days. + Dose of cells (2 × 10 5Upon further reduction of Fas DNR expression in T cells, Fas DNR-modified T cells continued to provide long-term survival in 100% of treated mice, whereas control-modified T cells lost efficacy overall (Figure 10G, right). Previous reports have demonstrated that 4-1BB-containing second-generation CARs express higher levels of antiapoptotic proteins compared to CARs containing the CD28 domain (80). These data in solid tumor B16 melanoma and blood E2a-PBX leukemia models indicate that Fas DNR expression in adoptively transferred T cells resulted in superior in vivo cell persistence and antitumor efficacy, regardless of whether the antigen-targeting construct was a TCR or 28ζ CAR. Example 3 FasDNR protects cells from FasL-induced apoptosis and does not affect T cell tumor-targeting function method

[0288] Cell Culture. Platinum-GP retroviral packaging cells (Cell Biolabs) were cultured in RPMI supplemented with 10% fetal bovine serum, 10 mM HEPES (Gibco) and 25 units / ml PenStrep (Gibco). Primary T cells were cultured in RPMI supplemented with 10% heat-inactivated human serum, 25 mM HEPES (Gibco) and 50 units / ml PenStrep (Gibco).

[0289] Isolation and expansion of human T cells. Buffy coats were obtained from healthy donors at the New York Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphocyte Separation Medium (Corning). CD8 + T cells were isolated using EasySep human CD8 + T cells were isolated using a T cell isolation kit (Stemcell). +T cells were activated with 5 μg / ml anti-CD3 (Miltenyi Biotec) antibody-coated plate and 1 μg / ml soluble anti-CD28 (Miltenyi Biotec). For viral transduction, T cells were treated with 50 IU / ml IL-2 (PeproTech) for 2 days prior to transduction.

[0290] Plasmid design and viral transduction. All plasmids for viral packaging were designed based on SFGγ retroviral vector. Feline endogenous retroviral envelope RD114 was used for co-transfection with SFGγ vector in platinum-GP cells. Lipofectamine 3000 (ThermoFisher) was used for co-transfection of platinum-GP cells. Primary T cells were transduced with viral supernatant on Retronectin (Takara) coated plates. Briefly, plates were coated with 20ug / ml Retronectin overnight at 4°C, then blocked with PBS with 2% FBS for 30 minutes at room temperature. Plates were washed with PBS and viral supernatant was loaded. Centrifugation was performed at 2000g and 32°C for 2 hours. Supernatant was aspirated and cells were loaded into each well. Plates were centrifuged again at 1200rpm, 32°C for 5 minutes, and incubated at 37°C for 2 days.

[0291] Flow cytometry and intracellular staining. The conjugated antibodies used for flow cytometry include Brilliant Violet 421™ anti-human EGFR (AY13, Biolegend), PE / Cy5 anti-human CD95 Fas (DX2, Biolegend), APC / Cyanine 7 anti-human CD95 Fas (DX2, Biolegend), PerCP / Cyanine 5.5 anti-human TNF-α (Mab11, Biolegend). For NY-ESO targeting TCR, PE anti-TCR Vβ13.1 (IMMU 222, Beckman Coulter) was used. For CAR staining, Alexa Fluor 647 AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 antibody (Jackson ImmunoResearch) was used.

[0292] FasL apoptosis assay. A form of soluble FasL oligomerized with a leucine zipper motif (FasL-LZ) was used at 100 ng / ml for all apoptosis assays. Cells were treated with FasL-LZ at 37°C for the designated time points. Cells were washed and stained for surface antibodies. Cells were stained with CellEvent™ Caspase-3 / 7 Green Detection Reagent (ThermoFisher) in FACS buffer for 25 min at 37°C and washed twice. Cells were then stained with APC Annexin V (Biolegend) in Annexin V binding buffer (Biolegend) for 25 min at room temperature. Cells were washed twice and resuspended in Annexin V binding buffer for flow cytometry.

[0293] Statistical analysis. All statistical analyses were performed using Prism 7 (GraphPad) software. No statistical methods were used to predetermine sample size. All analyses were performed on triplicate samples. Statistical comparisons between two groups were calculated by paired Student's t-test for matched samples. P<0.05 was considered statistically significant. result

[0294] The functionality of T cells engineered with Fas DNR and antigen recognition receptors (both TCR and CAR) was also evaluated. Multiple constructs were designed, as shown in Figure 17A. The resulting engineered human primary T cells express Fas, which protects T cells from FasL-induced apoptosis. DNR The cells expressed a T cell receptor (TCR) that targets the NY-ESO1 antigen, and EGFRt, which can be targeted by monoclonal antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (Figure 17B). DNR After antigen stimulation, both control and FasDNR cells showed increased TNFα staining (Fig. 17D). Furthermore, after exposure to FasL leucine zipper (FasL-lz) at different time points, Fas DNR T cells expressing IL-1 showed reduced staining for apoptotic markers.

[0295] Similarly, T cell functionality was assessed by the expression of Fas DNR , evaluated after co-engineering of a traceable truncated EGFR and the antigen-specific CAR anti-CD19 (CD1928ζ) (Figures 18A and 18B). After exposure to FasL leucine zipper (lz-FasL), Fas DNR T cells expressing the anti-CD19 CAR were protected from apoptosis, independent of expression of the anti-CD19 CAR (Figure 18C). Furthermore, T cells expressing only the anti-CD19 CAR (1928ζ) or Fas were protected from apoptosis after co-incubation with K562 cells expressing CD19. DNR Combination with (tEGFR-hFASDNR+CD1928ζ) showed comparable antigen-specific cytokine release and degranulation (Figure 18D). DNR reduces FasL-induced apoptosis without altering T cell function. References [ka] [ka] [ka] [ka] [ka] [ka] [ka] Embodiments of the Subject Matter of the Disclosure

[0296] From the foregoing description, it will be apparent that variations and modifications may be made to the subject matter of the present disclosure to adapt it to various uses and conditions, such embodiments still falling within the scope of the following claims.

[0297] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0298] 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

1. (a) a chimeric antigen receptor (CAR) that binds to a tumor antigen, and (b) an exogenous dominant-negative Fas polypeptide; A T cell comprising: the exogenous dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain of human Fas; and / or the modification comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence set forth in SEQ ID NO:10; and / or A T cell, wherein the modification comprises a point mutation at position 260 of human Fas having the amino acid sequence set forth in SEQ ID NO:

10.

2. The T cell of claim 1 , wherein the at least one modification in the cytoplasmic death domain prevents binding between the dominant negative Fas polypeptide and a FADD polypeptide.

3. The T cell of claim 1 or 2, wherein the dominant negative Fas polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

12.

4. The T cell of claim 3 , wherein the dominant negative Fas polypeptide has the amino acid sequence set forth in SEQ ID NO:

12.

5. The T cell of any one of claims 1 to 4, wherein the point mutation is D260V.

6. The T cell of claim 4, wherein the dominant negative Fas polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:

14.

7. The T cell of claim 6 , wherein the dominant negative Fas polypeptide has the amino acid sequence set forth in SEQ ID NO:

14.

8. The T cell of any one of claims 1 to 7, wherein the exogenous dominant-negative Fas polypeptide enhances cellular persistence of an immunoresponsive cell.

9. The T cell of any one of claims 1 to 8, wherein the exogenous dominant-negative Fas polypeptide reduces apoptosis or anergy of an immunoresponsive cell.

10. 10. The T cell of any one of claims 1 to 9, wherein the chimeric antigen receptor (CAR) is exogenous.

11. 11. The T cell of any one of claims 1 to 10, wherein the chimeric antigen receptor (CAR) is recombinantly expressed.

12. 12. The T cell of any one of claims 1 to 11, wherein the chimeric antigen receptor (CAR) is expressed from a vector.

13. The T cell of any one of claims 1 to 12, wherein the exogenous dominant negative Fas polypeptide is expressed from a vector.

14. The T cell of any one of claims 1 to 13, wherein the T cell is a cytotoxic T lymphocyte (CTL), a regulatory T cell or a natural killer T (NKT) cell.

15. 15. The T cell of any one of claims 1 to 14, which is autologous or allogeneic to the intended recipient.

16. The tumor antigen is CD19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K light chain, KDR, mutant KRAS, mutant PIK3CA, mutant IDH, mutant p53, mutant NRAS, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV 16. The T cell of any one of claims 1 to 15, wherein the T cell is selected from the group consisting of E6 oncoprotein, HPV E7 oncoprotein, and ERBB.

17. The T cell of claim 16, wherein the tumor antigen is CD19.

18. 18. The T cell of any one of claims 1 to 17, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain.

19. The T cell of claim 18, wherein the intracellular signaling domain further comprises at least one costimulatory signaling domain.

20. The T cell of claim 19, wherein the at least one costimulatory signaling domain comprises a CD28 polypeptide.

21. 21. The T cell of any one of claims 1 to 20, further comprising a suicide gene.

22. 22. The T cell of claim 21, wherein the suicide gene is herpes simplex virus thymidine kinase (hsv-tk), inducible caspase-9 suicide gene (iCasp-9), or a truncated human epidermal growth factor receptor (EGFRt) polypeptide.

23. 23. A composition comprising an effective amount of a T cell according to any one of claims 1 to 22.

24. 24. The composition of claim 23, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient.

25. 25. The composition according to claim 23 or 24, for treating and / or preventing neoplasia.

26. 26. A medicament for inducing and / or enhancing an immune response against a target antigen, comprising an effective amount of a T cell according to any one of claims 1 to 22 or a composition according to any one of claims 24 or 25.

27. 26. A medicament for reducing tumor burden in a subject, comprising an effective amount of a T cell according to any one of claims 1 to 22 or a composition according to any one of claims 24 or 25.

28. 28. The method of claim 27, wherein reducing tumor burden is reducing the number of tumor cells, reducing tumor size, and / or eradicating tumors in the subject.

29. 26. A medicament for treating and / or preventing neoplasia, comprising an effective amount of a T cell according to any one of claims 1 to 22 or a composition according to any one of claims 24 or 25.

30. 26. A medicament for prolonging the survival of a subject having a neoplasia, comprising an effective amount of a T cell according to any one of claims 1 to 22 or a composition according to any one of claims 24 or 25.

31. The pharmaceutical agent of any one of claims 27 to 30, wherein the tumor or neoplasm is selected from the group consisting of blood cancer, B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, myeloid leukemia and myelodysplastic syndrome (MDS).

32. 32. The method of claim 31, wherein the neoplasm is B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia or non-Hodgkin's lymphoma and the antigen is CD19.

33. 1. A medicament for treating a hematological cancer in a subject in need thereof, comprising an effective amount of a T cell, the T cell comprising a chimeric antigen receptor (CAR) that binds to a tumor antigen, and an exogenous dominant-negative Fas polypeptide; the exogenous dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain of human Fas; and / or the modification comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence set forth in SEQ ID NO:10; and / or The pharmaceutical, wherein the modification comprises a point mutation at position 260 of human Fas having the amino acid sequence shown in SEQ ID NO:

10.

34. The blood cancer is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), The pharmaceutical agent of claim 33, which is selected from the group consisting of chronic lymphocytic leukemia, non-Hodgkin's lymphoma, myeloid leukemia and myelodysplastic syndrome (MDS).

35. A medicament for treating a solid tumor in a subject in need thereof, comprising an effective amount of a T cell, the T cell comprising a chimeric antigen receptor (CAR) that binds to a tumor antigen, and an exogenous dominant-negative Fas polypeptide; the exogenous dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain of human Fas; and / or the modification comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence set forth in SEQ ID NO:10; and / or The pharmaceutical, wherein the modification comprises a point mutation at position 260 of human Fas having the amino acid sequence shown in SEQ ID NO:

10.

36. 36. The method of claim 35, wherein the solid tumor is a tumor of brain, breast, lung, gastrointestinal tract (including esophagus, stomach, small intestine, large intestine and rectum), pancreas, prostate, soft tissue / bone, uterus, cervix, ovary, kidney, skin, thymus, testis, head and neck, or liver origin.

37. A method for generating antigen-specific T cells in vitro, comprising introducing into a cell (a) a first nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a tumor antigen, and (b) a second nucleic acid encoding an exogenous dominant-negative Fas polypeptide; the exogenous dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain of human Fas; and / or the modification comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence set forth in SEQ ID NO:10; and / or The method, wherein the modification comprises a point mutation at position 260 of human Fas having the amino acid sequence set forth in SEQ ID NO:

10.

38. 38. The method of claim 37, wherein one or both of the first and second nucleic acids are operably linked to a promoter element.

39. 39. The method of claim 37 or 38, wherein one or both of the first and second nucleic acids are contained in a vector.

40. 40. The method of claim 39, wherein the vector is a retroviral vector.

41. (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds to a tumor antigen, and (b) a second nucleic acid sequence encoding an exogenous dominant-negative Fas polypeptide; the exogenous dominant-negative Fas polypeptide comprises at least one modification in the cytoplasmic death domain of human Fas; and / or the modification comprises a deletion of amino acids 230-314 of human Fas having the amino acid sequence set forth in SEQ ID NO:10; and / or A nucleic acid composition for generating recombinant T cells, wherein the modification comprises a point mutation at position 260 of human Fas having the amino acid sequence shown in SEQ ID NO:

10.

42. 42. The nucleic acid composition of claim 41, wherein one or both of the first and second nucleic acid sequences are operably linked to a promoter element.

43. 43. The nucleic acid composition of claim 41 or 42, wherein one or both of the first and second nucleic acid sequences are contained in a vector.

44. 44. The nucleic acid composition of claim 43, wherein the vector is a retroviral vector.

45. 45. A vector comprising the nucleic acid composition of any one of claims 41 to 44.

46. A kit for treating and / or preventing neoplasia or pathogen infection comprising a T cell according to any one of claims 1 to 22, a composition according to any one of claims 23 to 25, a nucleic acid composition according to any one of claims 41 to 44, or a vector according to claim 45.

47. 47. The kit of claim 46, further comprising written instructions for treating and / or preventing a neoplasia or pathogen infection.

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