Cells expressing the c-Kit mutation and their use

Abstract

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

Description

[Technical Field]

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

[0002] Sequence List This application is filed in ASCII format via EFS-Web and includes a sequence listing which is incorporated herein by reference in its entirety. The ASCII copy, created on 3 December 2020, is named 0727341174_ST25 and is 57,790 bytes in size.

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

[0004] 2. Background of the Invention Cell-based immunotherapy is a treatment with curative potential for treating cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material that encodes synthetic receptors for antigens, called innate or modified T cell receptors (TCRs) or chimeric antigen receptors (CARs), which are specific to selected antigens. Patient-engineered CAR T cells have demonstrated remarkable efficacy against a variety of humoral and solid malignancies.

[0005] Caribouid-mediated angioplasty (CARs) in clinical and preclinical development primarily utilize co-stimulatory domains, e.g., CD28 or 4-1BB. The persistence, particularly functional persistence, of these CARs has been shown to be associated with better outcomes. However, the demand for improved CARs with enhanced proliferation and persistence, as well as / or improved efficiency and activity, compared to existing CARs, without co-stimulatory domains, remains unmet. [Overview of the Initiative] [Means for solving the problem]

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

[0007] In certain embodiments, c-Kit is human c-Kit. In certain embodiments, the activating mutation is located within the intracellular domain of human c-Kit. In certain embodiments, the intracellular domain contains amino acids 544-977 of human c-Kit.

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

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

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

[0011] In certain embodiments, human c-Kit contains or consists of the amino acid sequence shown in Sequence ID No. 1.

[0012] In certain embodiments, the variant includes or consists of the amino acid sequence or portion thereof shown in SEQ ID NO: 2. In certain embodiments, the variant consists of amino acids 543-976 of SEQ ID NO: 2.

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

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

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

[0016] In certain embodiments, the cells are immune-responsive cells. In certain embodiments, the cells are lymphoid or myeloid cells. In certain embodiments, the lymphoid cells are selected from T cells, B cells, natural killer (NK) cells, and dendritic cells. In certain embodiments, the cells are T cells. In certain embodiments, the T cells are cytotoxic T lymphocytes (CTLs), γδT cells, tumor-infiltrating lymphocytes (TILs), regulatory T cells, or natural killer T (NKT) cells.

[0017] In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is mesothelin, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, K-light chain, KDR, LeY, L1 cell The antigen is selected from the group consisting of adhesion molecules, MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, oncoemetic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. In certain embodiments, the antigen is mesothelin.

[0018] In certain embodiments, the antigen-recognition receptor is selected from T cell receptors (TCRs), chimeric antigen receptors (CARs), and TCR-like fusion molecules. 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 intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region. In certain embodiments, the at least one co-stimulatory signaling region comprises a CD28 polypeptide. In certain embodiments, the CAR does not contain a co-stimulatory signaling region.

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

[0020] Furthermore, this disclosure provides compositions comprising: a) a variant of human c-Kit containing an activating mutation; and b) an antigen-recognizing receptor that binds to an antigen. In certain embodiments, the c-Kit variant is operably linked to a first promoter. In certain embodiments, the antigen-recognizing receptor is operably linked to a second promoter. In certain embodiments, one or both of the first and second promoters are inducible promoters. In certain embodiments, the inducible promoter is selected from the NFAT transcriptional response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter. This disclosure further provides cells comprising the compositions disclosed herein.

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

[0022] This disclosure further provides vectors comprising the nucleic acid compositions disclosed herein. This disclosure further provides cells comprising the vectors disclosed herein.

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

[0024] This disclosure further provides methods for reducing tumor burden in a subject. This disclosure also provides methods comprising the step of administering to a subject cells or pharmaceutical compositions disclosed herein. In certain embodiments, the methods reduce the number of tumor cells, decrease the tumor size, and / or eradicate the tumor in the subject.

[0025] This disclosure further provides methods for treating and / or preventing neoplasms. In certain embodiments, the method includes the step of administering to a subject cells or pharmaceutical compositions disclosed herein.

[0026] This disclosure further provides a method for extending the survival time of subjects having neoplasms. In certain embodiments, the method includes the step of administering to a subject cells or pharmaceutical compositions disclosed herein.

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

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

[0029] Furthermore, this disclosure provides kits comprising cells disclosed herein, compositions disclosed herein, nucleic acid compositions disclosed herein, or vectors disclosed herein. In certain embodiments, the kit further includes written instructions for treating and / or preventing neoplasms, pathogen infections, or infectious diseases. In embodiments of the present invention, for example, the following items are provided. (Item 1) (a) an antigen-recognition receptor that binds to an antigen, and (b) c-Kit variants containing activating mutations Cells containing this substance. (Item 2) The cells described in item 1, wherein the c-Kit is human c-Kit. (Item 3) The cells described in item 2, wherein the aforementioned activating mutation is located within the intracellular region of human c-Kit. (Item 4) The cell described in item 3, wherein the intracellular region contains amino acids 544-977 of human c-Kit. (Item 5) The cell described in any one of items 2 to 4, wherein the aforementioned activating mutation is located within amino acids 816-826 of human c-Kit. (Item 6) The cell described in any one of items 2 to 5, wherein the activating mutation is located at amino acid position 816 or amino acid position 822. (Item 7) The cell described in any one of items 2 to 4, wherein the activating mutation is located within amino acids 550-570 of human c-Kit. (Item 8) The cell described in any one of items 2 to 4 and 7, wherein the activating mutation is located at the 560th amino acid position of human c-Kit. (Item 9) The cells described in items 2 to 4, wherein the activating mutation is selected from D816V, D816Y, D816H, D816F, N822K, V560G, or a combination thereof. (Item 10) The cells according to any one of items 2 to 4 and 9, wherein the activating mutation includes or consists of D816V. (Item 11) The cell according to any one of items 2 to 10, wherein the human c-Kit contains or consists of the amino acid sequence shown in Sequence ID No. 1. (Item 12) The cell according to any one of items 1 to 11, wherein the mutant comprises or consists of the amino acid sequence or portion thereof shown in Sequence ID No. 2. (Item 13) The aforementioned mutant is a cell according to any one of items 1 to 12, wherein the mutant consists of amino acids 543 to 976 of SEQ ID NO: 2. (Item 14) The cell according to any one of items 1 to 13, wherein the c-Kit mutant is operably linked to an inducible promoter. (Item 15) The cells described in item 14, wherein the inducible promoter is selected from activated T cell nuclear factor (NFAT), transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter. (Item 16) The cell according to any one of items 1 to 15, wherein the c-Kit variant enhances the cell persistence of the cell. (Item 17) The cells according to any one of items 1 to 16, wherein the c-Kit variant reduces apoptosis or anergy in the cells. (Item 18) A cell according to any one of items 1 to 17, wherein the antigen recognition receptor is exogenous or endogenous. (Item 19) The cells described in any one of items 1 to 18, wherein the antigen recognition receptor is recombinantly expressed. (Item 20) The cell described in any one of items 1 to 19, wherein the antigen recognition receptor is expressed from the vector. (Item 21) The cells described in any one of items 1 to 20, wherein the c-Kit mutant is expressed from the vector. (Item 22) An immune-responsive cell, as described in any one of items 1 through 21. (Item 23) A cell that is either a lymphoid cell or a myeloid cell, as described in any one of items 1 through 22. (Item 24) The cells described in item 23, wherein the lymphoid cells are selected from T cells, B cells, natural killer (NK) cells, and dendritic cells. (Item 25) A T cell, as described in any one of items 1 through 24. (Item 26) The cells described in item 24 or 25, wherein the T cells are cytotoxic T lymphocytes (CTLs), γδT cells, tumor-infiltrating lymphocytes (TILs), regulatory T cells, or natural killer T (NKT) cells. (Item 27) A cell according to any one of items 1 to 26, wherein the antigen is a tumor antigen or a pathogen antigen. (Item 28) A cell according to any one of items 1 to 27, wherein the aforementioned antigen is a tumor antigen. (Item 29) The aforementioned tumor antigens include mesothelin, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, K-light chain, KDR, LeY, L1 cell adhesion molecule. Cells as described in item 28, selected from the group consisting of MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, tumor embryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB. (Item 30) The cells described in item 29, wherein the antigen is mesothelin. (Item 31) The cell according to any one of items 1 to 30, wherein the antigen recognition receptor is selected from T cell receptors (TCRs), chimeric antigen receptors (CARs), and TCR-like fusion molecules. (Item 32) A cell according to any one of items 1 to 31, wherein the antigen recognition receptor is a CAR. (Item 33) The cell described in item 32, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. (Item 34) The cell according to item 33, wherein the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region. (Item 35) The cell according to item 34, wherein at least one of the co-stimulatory signaling regions contains a CD28 polypeptide. (Item 36) The cell described in item 33, wherein the CAR does not contain a co-stimulatory signaling region. (Item 37) A method for generating antigen-specific immune-responsive cells, comprising the step of introducing into cells (a) a first nucleic acid sequence encoding an antigen-recognizing receptor that binds to an antigen; and (b) a second nucleic acid sequence encoding a c-Kit variant containing an activating mutation. (Item 38) The method according to item 37, wherein the first nucleic acid sequence is operably linked to a first promoter. (Item 39) The method according to item 37 or 38, wherein the second nucleic acid sequence is operably linked to a second promoter. (Item 40) The method according to any one of items 37 to 39, wherein one or both of the first and second nucleic acid sequences are included in the vector. (Item 41) The method according to item 341, wherein the vector is a retroviral vector. (Item 42) a) a variant of human c-Kit containing an activating mutation; and b) a composition comprising an antigen-recognizing receptor that binds to an antigen. (Item 43) The composition according to item 42, wherein the c-Kit variant is operably linked to a first promoter. (Item 44) The composition according to item 42 or 43, wherein the antigen recognition receptor is operably linked to a second promoter. (Item 45) The composition according to item 43 or 44, wherein one or both of the first and second promoters are inducible promoters. (Item 46) The composition according to item 45, wherein the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter. (Item 47) A nucleic acid composition comprising (a) a first polynucleotide encoding an antigen recognition receptor and (b) a second polynucleotide encoding a variant of human c-Kit containing an activating mutation. (Item 48) The nucleic acid composition according to item 47, further comprising a first promoter operably linked to the c-Kit variant. (Item 49) The nucleic acid composition according to item 47 or 48, further comprising a second promoter operably linked to the antigen recognition receptor. (Item 50) The nucleic acid composition according to item 48 or 49, wherein one or both of the first and second promoters are inducible promoters. (Item 51) The nucleic acid composition according to item 50, wherein the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter. (Item 52) A nucleic acid composition according to any one of items 47 to 51, wherein one or both of the first and second polynucleotides are contained in the vector. (Item 53) The nucleic acid composition according to item 52, wherein the vector is a retroviral vector. (Item 54) A vector comprising a nucleic acid composition as described in any one of items 47 to 53. (Item 55) Cells comprising a composition according to any one of items 42 to 46, a nucleic acid composition according to any one of items 47 to 53, or a vector according to item 54. (Item 56) A pharmaceutical composition comprising cells as described in any one of items 1 to 36 and 55, and a pharmaceutically acceptable excipient. (Item 57) The pharmaceutical composition according to item 56, further comprising a c-Kit inhibitor. (Item 58) The pharmaceutical composition according to item 57, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof. (Item 59) A pharmaceutical composition according to any one of items 56 to 58 for treating and / or preventing neoplasms, pathogen infections or infectious diseases. (Item 60) A method for reducing tumor burden in a subject, comprising the step of administering to the subject cells described in any one of items 1 to 36 and 55 or a pharmaceutical composition described in any one of items 56 to 59. (Item 61) The method according to item 60, wherein in the subject, the number of tumor cells is reduced, the tumor size is reduced, and / or the tumor is eradicated. (Item 62) A method for treating and / or preventing a neoplasm, comprising the step of administering to the subject a cell described in any one of items 1 to 36 and 55 or a pharmaceutical composition described in any one of items 56 to 59. (Item 63) A method for extending the survival time of a subject having a neoplasm, comprising the step of administering to the subject cells described in any one of items 1 to 36 and 55 or a pharmaceutical composition described in any one of items 56 to 59. (Item 64) The method according to any one of items 60 to 63, wherein the tumor or neoplasm is a solid tumor. (Item 65) The method according to item 64, wherein the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, bile duct cancer, and combinations thereof. (Item 66) The method according to item 64 or 65, wherein the solid tumor is a mesothelioma. (Item 67) The method according to item 64 or 65, wherein the solid tumor is lung cancer. (Item 68) The method according to any one of items 50 to 67, further comprising the step of administering a c-Kit inhibitor. (Item 69) The method according to item 68, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof. (Item 70) A kit comprising cells as described in any one of items 1 to 36 and 55, a composition as described in any one of items 42 to 46, a nucleic acid composition as described in any one of items 47 to 53, or a vector as described in item 54. (Item 71) The kit described in item 70, further including written instructions for treating and / or preventing neoplasms, pathogen infections, or infectious diseases.

[0030] 4. Brief explanation of the drawing The following detailed description, provided as an example but not intended to limit the subject matter of this disclosure to the specific embodiments described, can be understood in conjunction with the accompanying drawings. [Brief explanation of the drawing]

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

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

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

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

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

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

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

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

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

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

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

[0042] [Figure 12-1]Figures 12A–12D show the in vivo efficacy of M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells against low-MSLN lung tumors. Mice with established low-MSLN A549G lung tumors were treated with a single dose of 1 × 10⁵ T cells containing M28z, Mz-KITv, or M28z-KITv. UT represents the untreated control. Figure 12A shows the results for UT. Figure 12B shows the results for M28z CAR T cells. Figure 12C shows the results for M28z-KITv CAR T cells. Figure 12D shows the results for Mz-KITv CAR T cells. [Figure 12-2] Figures 12A–12D show the in vivo efficacy of M28z CAR T cells, Mz-KITv CAR T cells, or M28z-KITv CAR T cells against low-MSLN lung tumors. Mice with established low-MSLN A549G lung tumors were treated with a single dose of 1 × 10⁵ T cells containing M28z, Mz-KITv, or M28z-KITv. UT represents the untreated control. Figure 12A shows the results for UT. Figure 12B shows the results for M28z CAR T cells. Figure 12C shows the results for M28z-KITv CAR T cells. Figure 12D shows the results for Mz-KITv CAR T cells.

[0043] [Figure 13-1] Figures 13A–13D show the in vivo efficacy of T cells containing M28z, Mz-KITv, or M28z-KITv against high-MSLN lung tumors. Mice with established high-MSLN A549GM lung tumors were treated with a single dose of 1 × 10⁵ T cells containing M28z, Mz-KITv, or M28z-KITv. UT represents the untreated control. Figure 13A shows the results for UT. Figure 13B shows the results for M28z CAR T cells. Figure 13C shows the results for M28z-KITv CAR T cells. Figure 13D shows the results for Mz-KITv CAR T cells. [Figure 13-2]Figures 13A–13D show the in vivo efficacy of T cells containing M28z, Mz-KITv, or M28z-KITv against high-MSLN lung tumors. Mice with established high-MSLN A549GM lung tumors were treated with a single dose of 1 × 10⁵ T cells containing M28z, Mz-KITv, or M28z-KITv. UT represents the untreated control. Figure 13A shows the results for UT. Figure 13B shows the results for M28z CAR T cells. Figure 13C shows the results for M28z-KITv CAR T cells. Figure 13D shows the results for Mz-KITv CAR T cells.

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

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

[0046] [Figure 16-1] Figures 16A–16C show the antitumor activity of M28z, Mz-KITv, or M28z-KITv CAR T cells against high-MSLN-expressing lung tumors. NSG mice with established high-MSLN A549GM lung tumors were treated with a single dose of 1 × 10⁵ M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: Untreated control. Figure 16A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 16B and 16C show Kaplan-Meier survival analysis of mice, demonstrating the in vivo efficacy of IV administration of different CAR T cells. *, P<0.05. ***, P<0.001. [Figure 16-2] Figures 16A–16C show the antitumor activity of M28z, Mz-KITv, or M28z-KITv CAR T cells against high-MSLN-expressing lung tumors. NSG mice with established high-MSLN A549GM lung tumors were treated with a single dose of 1 × 10⁵ M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: Untreated control. Figure 16A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 16B and 16C show Kaplan-Meier survival analysis of mice, demonstrating the in vivo efficacy of IV administration of different CAR T cells. *, P<0.05. ***, P<0.001. [Figure 16-3] Figures 16A–16C show the antitumor activity of M28z, Mz-KITv, or M28z-KITv CAR T cells against high-MSLN-expressing lung tumors. NSG mice with established high-MSLN A549GM lung tumors were treated with a single dose of 1 × 10⁵ M28z, M28z-KITv, or Mz-KITv CAR T cells. UT: Untreated control. Figure 16A shows the results of in vivo monitoring of tumor burden in mice using bioluminescent imaging (BLI). Figures 16B and 16C show Kaplan-Meier survival analysis of mice, demonstrating the in vivo efficacy of IV administration of different CAR T cells. *, P<0.05. ***, P<0.001.

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

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

[0049] [Figure 19-1] Figures 19A and 19B show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing mesothelioma. Figure 19A shows low or high MSLN protein expression in MSTO cells for the generation of MG-LM and MGM cells, respectively. Figure 19B shows treatment of NSG mice with established low-MSLN mesothelioma (MG-LM) with a single dose of 5 × 10⁴ P28z, M28z, M28z-KITv, or Mz-KITv CAR T cells. Kaplan-Meier survival analysis compared the in vivo efficacy of intrapleural administration of different CAR T cells. *, P<0.05. **, P<0.01. [Figure 19-2]Figures 19A and 19B show the antitumor activity of M28z, Mz-KITv, and M28z-KITv CAR T cells against low-MSLN-expressing mesothelioma. Figure 19A shows low or high MSLN protein expression in MSTO cells for the generation of MG-LM and MGM cells, respectively. Figure 19B shows treatment of NSG mice with established low-MSLN mesothelioma (MG-LM) with a single dose of 5 × 10⁴ P28z, M28z, M28z-KITv, or Mz-KITv CAR T cells. Kaplan-Meier survival analysis compared the in vivo efficacy of intrapleural administration of different CAR T cells. *, P<0.05. **, P<0.01.

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

[0051] [Figure 21-1]Figures 21A and 21B show the analysis of PD1 expression in CAR T cells obtained from mice after exposure to high-mesothelin-expressing mesothelioma cells. Figure 21A shows the quantification of PD1 expression at different E:T ratios. Figure 21B shows flow cytometry graphs measuring PD1 expression at different E:T ratios. [Figure 21-2] Figures 21A and 21B show the analysis of PD1 expression in CAR T cells obtained from mice after exposure to high-mesothelin-expressing mesothelioma cells. Figure 21A shows the quantification of PD1 expression at different E:T ratios. Figure 21B shows flow cytometry graphs measuring PD1 expression at different E:T ratios.

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

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

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

[0055] Non-limiting embodiments of this disclosure are described herein and by examples.

[0056] For the purpose of clarifying, and not limiting, this disclosure, a more detailed explanation is divided into the following sub-sections: 5.1. Definition; 5.2. c-Kit variant; 5.3.Cell; 5.4. Antigen recognition receptors; 5.5. Programmed Death 1 in Dominant-Negative Form (PD-1 DN) 5.6. Compositions and vectors; 5.7. Polypeptides and analogs; 5.8. Administration; 5.9. Formulations; 5.10. Treatment methods; and 5.11. Kit

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

[0058] As used herein, the terms “about” or “approximately” mean a range of error that is permissible for a particular value as determined by those skilled in the art, which depends in part on the method by which the value is measured or determined, i.e., on the limits of the measuring system. For example, “about” may mean within three or more standard deviations, depending on the practice of the art. Alternatively, “about” may mean a range of up to 20% of a given value, e.g., up to 10%, up to 5%, or up to 1%. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, e.g., within five times or up to two times.

[0059] "Immune-responsive cells" refer to cells, precursors, or offspring that function in the immune response.

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

[0061] "Stimulating immune-responsive cells" means a signal that elicits a potent and sustained immune response. In various embodiments, this occurs either after the activation of immune cells (e.g., T cells) or simultaneously mediated via receptors including, but not limited to, CD28, CD137(4-1BB), OX40, CD40, and ICOS. Administering multiple stimulating signals can be crucial for initiating a potent and prolonged T-cell-mediated immune response. T cells may be immediately inhibited and rendered unable to respond to antigens. While the actions of these co-stimulatory signals can vary, they generally result in increased gene expression and the generation of long-lasting, proliferative, and anti-apoptotic T cells that respond strongly to antigens for complete and sustained eradication.

[0062] As used herein, the term “antigen-recognition receptor” refers to a receptor capable of activating immune or immune-responsive cells (e.g., T cells) in response to binding to an antigen.

[0063] As used herein, the term "antibody" also refers to not only intact antibody molecules but also fragments of antibody molecules that retain immunogenic 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" also refers to not only intact immunoglobulin molecules but also well-known active fragments F(ab’)2 and Fab. F(ab’)2 and Fab fragments lacking the Fe fragment of intact antibodies disappear more rapidly from circulation and may have less non-specific tissue binding than intact antibodies (Wahl et al., J Nucl Med (1983); 24:316-325). As used herein, antibodies include whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single-chain variable fragments (scFv), fusion polypeptides and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H ), and a heavy chain constant region (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 (abbreviated herein as V L ), and a light chain constant C L region. The light chain constant region is composed of one domain, C L . The V H region and the V L region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR). Each V H and V LIt consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl 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 the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including the first component (C1q) of the classical complement system.

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

[0065] As used herein, the terms “single-chain variable fragment” or “scFv” mean V H ::V L The heavy chain of immunoglobulins (V) is covalently linked to form a heterodimer. H ) and light chain (V L It is a fusion protein of the variable region of ). H and V LIt is either directly linked or linked by a linker encoding a peptide (e.g., 10, 15, 20, or 25 amino acids), thereby V H The N-terminus is V L Connect to the C terminal of or V H The C-terminus is V L It connects to the N-terminus of . Linkers are typically glycine-rich with respect to flexibility and serine or threonine-rich with respect to solubility. As used herein, “linker” means a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids so that they are linked to one another. As used herein, “peptide linker” means a functional group (e.g., a nucleotide or polypeptide) that links two proteins together (e.g., V H Domain and V L Refers to one or more amino acids used to link the domain. In certain embodiments, the linker includes the sequence shown in SEQ ID NO: 16 provided below: GGGGSGGGGSGGGGS[Sequence No. 16]

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

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

[0068] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are described by Huston et al., Proc. Nat. Acad. Sci. USA (1988); 85:5879-5883). H and V L It may be expressed from nucleic acids containing coding sequences; see U.S. Patents No. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publications No. 20050196754 and 20050196754. Several inhibitory antagonists, scFv, have been described (Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle (2013); 4(1):79-86; Shieh et al., J Imunol (2009); 183(4):2277-85; Giomarelli et al., Thromb Haemost (2007); 97(6):955-63; Fife eta., JCI (2006); 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol (1995); 2(10):31-40). The scFv of agonists with stimulating activity has been described (Peter et al., J Biol Chem (2003); 25278(38): 36740-7; Xie et al., Nat Biotech (1997); 15(8): 768-71; Ledbetter et al., Crit Rev Immunol (1997); 17(5-6): 427-55; Ho et al., BioChem Biophys Acta (2003); 1638(3): 257-66).

[0069] As used herein, the term "affinity" means a measure of binding strength. Affinity may depend on the degree of stereochemical fit between the antibody combining site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "avidence," which refers to the strength of antigen-antibody binding after reversible complex formation. 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).

[0070] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to a molecule comprising an intracellular signaling domain capable of activating or stimulating immune response cells, and an extracellular antigen-binding domain fused to a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv may originate from fusion to the variable heavy and light chain regions of an antibody. Alternatively, the scFv may originate from a Fab (obtained, for example, from a Fab library, instead of from an antibody). In certain embodiments, the scFv fuses to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for an antigen.

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

[0072] Polynucleotides having "substantially identical" or "substantially homologous" a given endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" means a pair of complementary polynucleotide sequences (e.g., the genes described herein) or parts thereof that form a double-stranded molecule under various stringency conditions. (Wahl et al., Methods Enzymol. (1987); 152:399; Kimmel, Methods Enzymol. (1987); 152:507).

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

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

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

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

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

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

[0079] The terms “constitutive expression” or “constitutively expressed” as used herein refer to expression under all physiological conditions or being expressed under all physiological conditions.

[0080] "Disease" means any condition, disease, or disorder that damages or interferes with the normal functioning of a cell, tissue, or organ, such as neoplasms and pathogenic infections of cells.

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

[0082] "Enhancing tolerability" means preventing the activation of autoreactive or immune-responsive cells that target the transplanted organ or tissue.

[0083] "Endogenous" refers to nucleic acid molecules or polypeptides that are normally expressed in cells or tissues.

[0084] "Exogenous" means a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Therefore, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, including foreign, heterogeneous, overexpressed nucleic acid molecules and polypeptides. "Exogenous" nucleic acid means a nucleic acid that is not present in natural wild-type cells, and for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, location / situation, or both. For clarification, an exogenous nucleic acid may have the same or different sequence compared to its natural endogenous counterpart, may be introduced into the cell itself or its precursor by genetic engineering, and may be ligated to an alternative control sequence, such as a non-natural promoter or secretory sequence, as necessary.

[0085] "Heterogeneous nucleic acid molecules or polypeptides" means nucleic acid molecules (e.g., cDNA, DNA, or RNA molecules) or polypeptides that are not normally present in cells or samples obtained from cells. These nucleic acids may originate from another organism, or they may be mRNA molecules that are not normally expressed in cells or samples.

[0086] "Modulating" means changing something positively or negatively. Exemplary modulations include changes of approximately 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0087] "Increase" means to change positively by at least about 5%. The change may be about 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more.

[0088] "To decrease" means to change it negatively by at least about 5%. The change may be about 5%, 10%, 25%, 30%, 50%, 75%, or even up to about 100%.

[0089] The terms “isolated,” “purified,” or “biologically pure” refer to substances that do not contain, to varying degrees, the components that are normally present in their natural state. “Isolating” indicates the degree of separation from the original source or environment. “Purifying” indicates a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is substantially free of other substances such that none of the impurities substantially affect the protein’s biological properties and cause any other harmful consequences. That is, nucleic acids or peptides are purified if, when produced by recombinant DNA techniques, the cell material, viral material, and culture medium are substantially free of those materials, or if, when chemically synthesized, the chemical precursors and other chemicals are substantially free of those materials. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” may indicate that the nucleic acid or protein produces essentially one band on an electrophoretic gel. In proteins that can be modified, for example, phosphorylation or glycosylation, various modifications can occur in various isolated proteins, which can then be purified separately.

[0090] "Isolated cells" refers to cells that have been separated from the molecules and / or cellular components that naturally accompany them.

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

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

[0093] A "receptor" refers to a polypeptide or a portion thereof located on the cell membrane that selectively binds to one or more ligands.

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

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

[0096] "Secreted" refers to polypeptides released from a cell via secretory pathways such as the endoplasmic reticulum and Golgi apparatus, and as vesicles that transiently fuse in the cell plasma membrane and release proteins to the outside of the cell.

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

[0098] "Specifically binding" means a polypeptide or fragment thereof that recognizes and binds to a biomolecule of interest (e.g., a polypeptide), but substantially does not recognize or bind to other molecules in a sample that naturally contains the polypeptide of this disclosure, such as a biological sample.

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

[0100] As used herein, “treatment” refers to a therapeutic intervention that attempts to alter the course of a disease in the individual or cell being treated, and may be performed either preventively or during the course of a clinicopathological condition. The therapeutic effects of a treatment may include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improving prognosis. By preventing the progression of disease or disability, a treatment may prevent the worsening of a disability in an affected or diagnosed subject, or a subject suspected of having a disability, and a treatment may also prevent the onset or symptoms of a disability in a subject at risk of or suspected of having a disability.

[0101] "Individual" or "subject" as used herein means human or non-human animal, such as vertebrates including mammals. Mammals include, but are not limited to, humans, primates, farm animals, game animals, rodents and companion animals. Non-exclusive examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, and non-human primates such as apes and monkeys. The term "immunocompromised" as used herein means an immunocompromised subject. Subjects are highly susceptible to opportunistic infections, which are infections caused by organisms that do not normally cause disease in people with healthy immune systems but can affect people with poorly functioning or suppressed immune systems.

[0102] Other aspects of the subject matter of this disclosure are described in the following disclosures and are within the scope of the subject matter of this disclosure.

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

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

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

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

[0107] The cells of the subject of this disclosure include c-Kit variants. In certain embodiments, the c-Kit variant includes an activating mutation.

[0108] In certain embodiments, the activating mutation is a gain-of-function mutation. In certain embodiments, the activating mutation is a mutation that results in an enhanced effect in the gene product compared to a gene product without such a mutation (e.g., a wild-type protein).

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

[0110] Activating mutations in c-Kit promote proliferation and prevent apoptosis in cells containing c-Kit mutants. Activating mutations in c-Kit also make cells containing c-Kit mutants resistant to PD-L1 / 2-PD-1 inhibition.

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

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

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

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

Chemical Structure

Chemical Structure

[0115] 5.3. Cells The subject matter of this disclosure provides cells containing c-Kit variants disclosed herein (e.g., those disclosed in Section 5.2). In certain embodiments, the c-Kit variant is an exogenous c-Kit variant.

[0116] In certain embodiments, the cells are selected from lymphoid and myeloid cells. In certain embodiments, the cells are immune-responsive cells. In certain embodiments, the immune-responsive cells are lymphoid cells.

[0117] In certain embodiments, the cells are lymphoid cells. Lymphoid cells can be responsible for antibody production, regulation of the cellular immune system, detection of foreign virulence factors in the blood, and detection of foreign cells in the host. Non-limiting examples of lymphoid cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells that can differentiate into lymphoid cells. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells).

[0118] In certain embodiments, the cells are T cells. T cells may also be lymphocytes that mature in the thymus and are largely responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject of this 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 type 2 effector memory T cells: for example, T EM Cells and T EMRA T cells can be any type of T cell, including but not limited to CD4 cells, regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introducing antigen-recognizing receptors, such as CARs or TCRs. In certain embodiments, immune-responsive cells are T cells. T cells are CD4 cells.+ T cells or CD8 + It may be a T cell. In a particular embodiment, the T cell is a CD4 + These are T cells. In certain embodiments, T cells are CD8 + These are T cells.

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

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

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

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

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

[0124] In certain embodiments, the cells of this disclosure are capable of modulating the tumor microenvironment. Tumors have a microenvironment that is adversarial to the host immune response, involving a set of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This “adversarial tumor microenvironment” is invasive regulatory CD4 + This includes various immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and ligands targeted to immunosuppressive receptors (CTLA-4 and PD-1) expressed by activated T cells. While these immunosuppressive mechanisms play a role in maintaining tolerability and suppressing inappropriate immune responses, within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. Collectively, these immunosuppressive factors may induce either pronounced anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with targeted tumor cells.

[0125] In certain embodiments, the cells of the Disclosure have increased cell proliferation and / or cell persistence. In certain embodiments, the immune-responsive cells of the Disclosure have reduced apoptosis and / or anergy.

[0126] In certain embodiments, the cells further comprise an antigen-recognizing receptor (e.g., CAR or TCR) that binds to an antigen. The cells may be transduced with an antigen-recognizing receptor and c-Kit variant, which are exogenously activated to cause the cells to co-express the antigen-recognizing receptor and c-Kit variant.

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

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

[0129] 5.4.1. Antigens Antigen recognition receptors can bind to tumor antigens or pathogen antigens.

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

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

[0132] In certain embodiments, for example, an antigen recognition receptor for use in treating and / or preventing, for example, a pathogen infection or other infectious disease in an immunocompromised subject binds to a pathogen antigen. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protists that can cause disease.

[0133] Non-exclusive examples of viruses include Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 (also known 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); Fla viridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (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., Hantan virus, Bunyavirus (bunga (virus), phlebovirus and nairavirus; Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); as well as unclassified viruses (e.g., delta hepatitis factors (considered to be deficient satellites of hepatitis B virus), non-A non-B hepatitis factors (class 1 = internal infection, class 2 = parenteral infection (i.e., hepatitis C));Examples include Norwalk and related viruses, as well as astroviruses, human papillomavirus (i.e., HPV), JC virus, Epstein-Barr virus, and Merkel cell polyomavirus.

[0134] Non-specific examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, 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 (Viridance group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, and Bacillus. antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, clostridium difficile and Actinomyces israelli.

[0135] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus. 5.4.2. T cell receptor (TCR)

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

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

[0138] In certain embodiments, the TCR can form receptor complexes with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. When the TCR complex associates with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

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

[0140] In certain embodiments, the antigen-recognition receptor is an exogenous TCR. In certain embodiments, the antigen-recognition receptor is a recombinant TCR. In certain embodiments, the antigen-recognition receptor is a TCR that does not exist in nature. In certain embodiments, the TCR that does not exist in nature differs from any of the naturally occurring TCRs by at least one amino acid residue. In certain embodiments, the TCR that does not exist in nature differs from any of the naturally occurring TCRs 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 TCR that does not exist in nature is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, naturally occurring TCRs are modified from naturally occurring TCRs 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. 5.4.3. Chimeric Antigen Receptors (CARs)

[0141] In certain embodiments, the antigen-recognition receptor is a CAR. A CAR is an engineered receptor that is transplanted or conferred with desired specificity onto immune effector cells. By facilitating the transfer of the coding sequence using a retroviral vector, the specificity of a monoclonal antibody can be transplanted onto T cells using a CAR.

[0142] Three generations of CARs exist. "First-generation" CARs typically consist of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. "First-generation" CARs provide de novo antigen recognition and, independently of HLA-mediated antigen presentation, enable CD4 cellular signaling via their CD3ζ chain signaling domain in a single fusion molecule. + T cells and CD8 +Both activations of T cells can be achieved. "Second-generation" CARs provide further signaling to T cells by adding intracellular signaling domains derived from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic end of the CAR. "Second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include those that provide multiple co-stimulatory molecules (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen-recognition receptor is a first-generation CAR. In certain embodiments, the antigen-recognition receptor is a CAR that does not contain the intracellular signaling domain of a co-stimulatory molecule. In certain embodiments, the antigen-recognition receptor is a second-generation CAR.

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

[0144] 5.4.3.1. Extracellular antigen-binding domain of CAR In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen. In certain embodiments, the antigen is mesothelin. In certain embodiments, the extracellular antigen-binding domain is scFv. In certain embodiments, scFv is human scFv. In certain embodiments, scFv is humanized scFv. In certain embodiments, scFv is mouse scFv. In certain embodiments, the extracellular antigen-binding domain is crosslinked Fab as needed. In certain embodiments, the extracellular antigen-binding domain is F(ab)2. In certain embodiments, any of the above molecules can be contained within a heterologous sequence fusion protein to form an extracellular antigen-binding domain. In certain embodiments, scFv is identified by screening an scFv phage library using an antigen-Fc fusion protein.

[0145] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR (e.g., scFv or its analogues are embodied) is approximately 2 × 10⁻¹⁶ -7 M or a dissociation constant less than (K) d ) binds to the antigen. In a particular embodiment, K d It is approximately 2 x 10 -7 M or less, approximately 1 x 10 -7 M or smaller, approximately 9 x 10 -8 M or less, approximately 1 x 10 -8 M or smaller, approximately 9 x 10 -9 M or less, approximately 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, approximately 3 x 10 -9 Or less, approximately 2 x 10 -9 M or less, or approximately 1 × 10 -9 M or less. In certain non-limiting embodiments, K d It is approximately 3 x 10 -9 M or less. In certain non-limiting embodiments, K d It is approximately 1 x 10 -9 M ~ approx. 3×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1.5 × 10 -9 M ~ approx. 3×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1.5 × 10 -9 M ~ approx. 2.7×10 -7 It is M.

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

[0147] The binding of the extracellular antigen-binding domain of CAR (in embodiments, e.g., in scFv) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a specific target protein-antibody complex by using a reagent (e.g., antibody, or scFv) that is 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, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a γ counter or scintillation counter, or by autoradiography. In certain embodiments, the mesothelin-targeted extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, citrin, Venus, and YPet). In one embodiment, a mesothelin-targeted human scFv is labeled with GFP.

[0148] In certain non-limiting embodiments, the extracellular antigen-binding domain of CAR recognizes or binds to human mesothelin having approximately 1,000 or more mesothelin binding sites / cells. In certain embodiments, the extracellular antigen-binding domain of CAR recognizes or binds to human mesothelin having approximately 1,000 to approximately 50,000 mesothelin binding sites / cells. In some embodiments, the extracellular antigen-binding domain of CAR does not recognize or bind to human mesothelin having fewer than 1,000 mesothelin binding sites / cells, e.g., human mesothelin expressed in normal tissues, e.g., normal pleura, pericardium, and peritoneal tissue. In certain embodiments, the extracellular antigen-binding domain of CAR does not recognize or bind to human mesothelin having more than 50,000 mesothelin binding sites / cells. In certain embodiments, the human scFv contained in the CAR recognizes or binds to human mesothelin having approximately 1,000 to approximately 50,000 mesothelin binding sites / cell mesothelin expression levels. In certain embodiments, the human scFv contained in the CAR does not recognize or bind to human mesothelin having more than 50,000 or fewer than 1,000 mesothelin binding sites / cell mesothelin expression levels.

[0149] In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) includes the amino acid sequence shown in SEQ ID NO: 4 or a conserved modification thereof. H V, which includes the amino acid sequence shown in CDR1, SEQ ID NO: 5 or its conservation modifications. H CDR2 and the amino acid sequence shown in Sequence ID No. 6, including its conservation modifications. H Includes CDR3. In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) includes the amino acid sequence shown in SEQ ID NO: 4. H V containing the amino acid sequence shown in CDR1, SEQ ID NO: 5 H V containing the amino acid sequence shown in CDR2 and SEQ ID NO: 6 HIncludes CDR3. In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) includes the amino acid sequence shown in SEQ ID NO: 7 or a conserved modification thereof. L V, which includes the amino acid sequence shown in CDR1, SEQ ID NO: 8 or its conservation modification. L V, which includes the amino acid sequence shown in CDR2 and SEQ ID NO: 9 or its conservation modifications. L Includes CDR3. In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) contains the amino acid sequence shown in SEQ ID NO: 7. L CDR1 contains the amino acid sequence shown in SEQ ID NO: 8. L V, which contains the amino acid sequence shown in CDR2 and SEQ ID NO: 9 L Includes CDR3.

[0150] In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) includes the amino acid sequence shown in SEQ ID NO: 4 or a conserved modification thereof. H V, which includes the amino acid sequence shown in CDR1, SEQ ID NO: 5 or its conservation modifications. H CDR2, the amino acid sequence shown in Sequence ID No. 6, and its conservation modifications, including V H V, which includes the amino acid sequence shown in CDR3, SEQ ID NO: 7 or its conservation modification. L V, which includes the amino acid sequence shown in CDR1, SEQ ID NO: 8 or its conservation modification. L V, which includes the amino acid sequence shown in CDR2 and SEQ ID NO: 9 or its conservation modifications. L Contains CDR3. In certain embodiments, the extracellular antigen-binding domain contains amino acids having the sequence shown in SEQ ID NO: 4. H V containing the amino acid sequence shown in CDR1, SEQ ID NO: 5 H V containing the amino acid sequence shown in CDR2, SEQ ID NO: 6 H CDR3 contains the amino acid sequence shown in SEQ ID NO: 7. L CDR1 contains the amino acid sequence shown in SEQ ID NO: 8. L V, which contains the amino acid sequence shown in CDR2 and SEQ ID NO: 9 LIt includes CDR3. In certain embodiments, the CDRs are identified according to the Kabat numbering system.

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

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

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

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

[0155] In a particular embodiment, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 is shown in SEQ ID NO: 15.

[0156] In a particular embodiment, the scFv is a human scFv. Sequence IDs 4-15 are provided below: [ka] [ka]

[0157] As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the mesothelin-targeting CAR of this disclosure (e.g., the extracellular antigen-binding domain of the CAR), including the amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications may be introduced into the extracellular antigen-binding domain of the CAR of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid belonging to the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; and nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, one or more amino acid residues within the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retained function (i.e., the function shown in (c) to (l) above) using the functional assay described herein. In certain embodiments, one or fewer, two or fewer, three or fewer, four or five or fewer residues within a specified sequence or CDR region are modified.

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

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

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

[0161] Furthermore, the amino acid sequences of the subject matter of this disclosure can be used as "query sequences" to perform searches against public databases, for example, to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J. Mol. Biol. 215:403-10. The BLAST protein search can be performed using the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., the sequences of the heavy and light chain variable regions of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, the gapped BLAST described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402 can be used. When using BLAST and gapped BLAST programs, you can use the default parameters of each program (e.g., XBLAST and NBLAST).

[0162] 5.4.3.2. Transmembrane domain of CAR In certain non-limiting embodiments, the transmembrane domain of a CAR includes a hydrophobic alpha-helix extending over at least a portion of the membrane. Various transmembrane domains result in various receptor stabilities. After antigen recognition, the receptor clusters, and the signal is transmitted to the cell. According to the subject of this disclosure, the transmembrane domain of a CAR may include native or modified transmembrane domains of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, synthetic peptides (not based on proteins related to the immune response), or combinations thereof.

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

[0164] In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence or 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: 19) provided below (in this specification, homology can be determined using standard software such as BLAST or FASTA), and / or may optionally contain up to 1, or up to 2, or up to 3 conserved amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 19, having at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acid lengths. Alternatively, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO: 19. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or having amino acids 137-209 of SEQ ID NO: 19. [ka]

[0165] In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence or 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 (Sequence ID 20) provided below (in this specification, homology can be determined using standard software such as BLAST or FASTA), and / or may optionally contain up to 1, or up to 2, or up to 3 conserved amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a continuous portion of Sequence ID 20, having at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acid lengths. Alternatively, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 20. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or having amino acids 151-219 of SEQ ID NO: 20. [ka]

[0166] In a particular embodiment, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or having the amino acid sequence shown in SEQ ID NO: 21 provided below: STTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY[Sequence No. 21]

[0167] According to the subject matter of this disclosure, “CD8 nucleic acid molecule” refers to a polynucleotide that encodes a CD8 polypeptide.

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

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

[0170] The CD28 polypeptide comprises or consists of an amino acid sequence or 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 sequence having NCBI reference number P10747 or NP_006130 (SEQ ID NO: 2), and / or may optionally contain up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 23, having at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acid lengths. In various non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 153-179, or 200-220 of SEQ ID NO: 23. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide containing amino acids 153-179 of SEQ ID NO: 23, or the CD28 polypeptide comprising SEQ ID NO: 23. SEQ ID NO: 23 is provided below: [ka]

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

[0172] In a particular embodiment, an exemplary CD28 nucleic acid molecule encoding the CD28 polypeptide consisting of amino acids 153-179 of SEQ ID NO: 23 is shown in SEQ ID NO: 24 provided below. ttttgggtgctggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg[Sequence No. 24]

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

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

[0175] In certain non-limiting embodiments, the CAR further includes a spacer region that ligates an extracellular antigen-binding domain to a transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to be oriented in various directions to facilitate antigen recognition.

[0176] In certain non-limiting embodiments, the hinge / spacer region of CAR includes native or modified hinge regions of CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, synthetic polypeptides (not based on proteins related to immune responses), or combinations thereof. The hinge / spacer region may be a hinge region derived from IgG1, or a portion of the CH2CH3 region and CD3 of an immunoglobulin, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 23), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 19 or a portion of SEQ ID NO: 20), any of the aforementioned variants that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

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

[0178] In certain embodiments, the intracellular signaling domain of CAR contains native CD3ζ. In certain embodiments, the CD3ζ polypeptide contains or comprises an amino acid sequence or 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: 27), and / or may optionally contain up to 1, or up to 2, or up to 3 conserved amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide contains or comprises an amino acid sequence that is a contiguous portion of SEQ ID NO: 27, having at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acid lengths. Alternatively, in various non-limiting embodiments, the CD3ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 27. In certain embodiments, the intracellular signaling domain of CAR comprises a CD3ζ polypeptide comprising or having amino acids 52-164 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below: [ka]

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

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

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

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

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

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

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

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

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

[0188] In certain embodiments, the intracellular signaling domain of CAR includes a co-stimulatory signaling region comprising two co-stimulatory molecules or portions thereof (e.g., intracellular domains of co-stimulatory molecules), such as the intracellular domains of CD28 and 4-1BB, or the intracellular domains of CD28 and OX40.

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

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

[0191] In certain embodiments, the CAR is a mesothelin-targeted CAR. In certain embodiments, the CAR is (a) V consisting of the amino acid sequence shown in SEQ ID NO: 4H V, consisting of the amino acid sequence shown in CDR1, SEQ ID NO: 5 H V consists of the amino acid sequence shown in CDR2, SEQ ID NO: 6. H V, consisting of the amino acid sequence shown in CDR3, SEQ ID NO: 7 L V, consisting of the amino acid sequence shown in CDR1, SEQ ID NO: 8. L V consists of CDR2 and the amino acid sequence shown in SEQ ID NO: 9. L (b) an extracellular antigen-binding domain containing CDR3; (c) a transmembrane domain containing the transmembrane domain of CD8; and (d) an intracellular signaling domain containing the CD3ζ polypeptide, but without a co-stimulatory signaling region.

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

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

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

[0195] 5.5. Programmed Death 1 in Dominant-Negative Form (PD-1 DN) In certain embodiments, the cells of the subject of this disclosure further include programmed death 1 in a dominant-negative form (referred to as "PD-1 DN").

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

[0197] In certain embodiments, cells, such as T cells, are engineered to express dominant-negative (DN) PD-1.

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

[0199] Programmed cell death protein 1 (PD-1) is a negative immunomodulator of activated T cells, expressed on endogenous macrophages and dendritic cells, through binding to its corresponding ligands, PD-L1 and PD-L2. PD-1 is a 268-amino acid type I membrane protein. PD-1 consists of two ligands, PD-L1 and PD-L2, which are members of the B7 family. The protein structure includes an extracellular IgV domain followed by a transmembrane domain and an intracellular terminal. The intracellular terminal contains two phosphorylation sites located within an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif. PD-1 negatively modulates TCR signaling. SHP-1 and SHP-2 phosphatases bind to the cytoplasmic terminal of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism used by tumor cells to evade the host immune system. In preclinical and clinical trials, PD-1 blockade with antagonist antibodies induced an antitumor response mediated through the host endogenous immune system.

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

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

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

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

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

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

[0206] In a particular embodiment, PD-1 DN comprises or consists of amino acids 1 to 165 of SEQ ID NO: 33.

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

[0208] In certain embodiments, PD-1 DN comprises or consists of amino acids 21-151 of SEQ ID NO: 33. In certain embodiments, PD-1 DN comprises or consists of amino acids 1-151 of SEQ ID NO: 33. In certain embodiments, PD-1 DN comprises or consists of amino acids 21-151 of SEQ ID NO: 33. In certain embodiments, PD-1 DN comprises or consists of an amino acid sequence starting with amino acid 21 of SEQ ID NO: 33 and ending with amino acids between 151-165 of SEQ ID NO: 33.

[0209] In certain embodiments, the PD-1 DN further comprises the CD8 polypeptide. In certain embodiments, the PD-1 DN comprises the extracellular domain or a portion of the PD-1 (e.g., the extracellular ligand-binding domain) fused to the transmembrane domain and / or hinge domain of CD8. In certain embodiments, the PD-1 DN comprises the transmembrane domain of CD8 (e.g., amino acids 183-203 of SEQ ID NO: 19). Such embodiments represent a chimeric DN morphology comprising a transmembrane domain derived from a different (heterogeneous) polypeptide. As described above, a PD-1 DN comprising a heterogeneous domain, e.g., a transmembrane domain, may optionally contain further sequences derived from the heterogeneous polypeptide. In certain embodiments, the PD-1 DN comprises further sequences derived from the heterogeneous polypeptide at the N-terminal end of the transmembrane domain. In certain embodiments, the PD-1 DN comprises the hinge domain of CD8. In certain embodiments, the heterologous sequence includes a further N-terminal sequence of the CD8 polypeptide (e.g., amino acids 137-182 of SEQ ID NO: 19 (or, if necessary, starting at amino acids 138 or 139)). In certain embodiments, the PD-1 DN includes a further sequence derived from the heterologous polypeptide on the C-terminal side of the transmembrane domain of CD8. In certain embodiments, the further C-terminal sequence is amino acids 204-209 of SEQ ID NO: 19.

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

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

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

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

[0214] In a particular embodiment, PD-1 DN comprises the amino acid sequence shown in SEQ ID NO: 39 provided below. [ka] [ka]

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

[0216] In a particular embodiment, PD-1 DN comprises the amino acid sequence shown in SEQ ID NO: 41 provided below. [ka]

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

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

[0219] 5.6. Compositions and Vectors The subject matter of this disclosure provides (a) compositions comprising c-Kit variants disclosed herein (e.g., disclosed in item 5.2) and antigen recognition receptors disclosed herein (e.g., disclosed in item 5.4). Cells comprising such compositions are also provided.

[0220] In certain embodiments, the c-Kit variant is operably ligated to a first promoter. In certain embodiments, the antigen-recognition receptor is operably ligated to a second promoter.

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

[0222] In certain embodiments, the nucleic acid composition further comprises a first promoter operably ligated to a c-Kit variant. In certain embodiments, the nucleic acid composition further comprises a second promoter operably ligated to an antigen recognition receptor.

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

[0224] Compositions and nucleic acid compositions can be administered to a subject or / or delivered intracellularly by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transduction of recombinant DNA constructs into a substantially homogeneous cellular composition. In certain embodiments, retroviral vectors (e.g., either gamma retroviral vectors or lentiviral vectors) are used to introduce DNA constructs into cells. For example, polynucleotides encoding antigen-recognition receptors can be cloned into retroviral vectors, and expression can be driven from their endogenous promoter, from the long-chain terminal repeat sequence of the retrovirus, or from a promoter specific to the target cell type of interest. Nonviral vectors can be used in a similar manner.

[0225] Retroviral vectors are generally used for transduction for the initial genetic modification of cells to include antigen-recognition receptors (e.g., CARs or TCRs), however, any other suitable viral vectors or nonviral delivery systems can also be used. Antigen-recognition receptors and c-Kit variants can be constructed in a single multi-cistronic expression cassette, multiple expression cassettes in a single vector, or in multiple vectors. Examples of elements that generate polycistronic expression cassettes include, but are not limited to, various viral and nonviral intra-sequence ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, plague virus IRES, aphthous virulence virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). The combination of retroviral vectors and appropriate packaging systems is also suitable when the capsid protein is functional for infecting human cells. Various amphotropic virus-producing cell systems are known and include, but are not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. (1985); 5:431-437); PA317 (Miller, et al. Mol. Cell. Biol. (1986); 6:2895-2902); and CRIP (Danos, et al. Proc. Natl. Acad. Sci. USA (1988); 85:6460-6464). Non-amphotropic particles, such as those pseudotyped with VSVG, RD114, or GALV envelopes and any other known in the art, are also preferred.

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

[0227] Other transdependent viral vectors may be used to modify immune-responsive cells. In certain embodiments, the selected vectors exhibit high infection efficiency as well as 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 papillomavirus, or herpesvirus, such as Epstein-Barr virus (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, Examples include the vectors described in 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 are particularly well-developed and used in clinical practice (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0228] Non-viral approaches can also be used for genetic modification of immune-responsive cells. For example, by administering nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialorosomucoid-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, By 1990, nucleic acid molecules can be introduced into immune-responsive cells. Other nonviral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for the delivery of DNA into cells. Transplantation of normal genes into the affected tissue of target can be achieved by transferring normal nucleic acids into ex vivo cultureable cell types (e.g., autologous or xenogeneic primary cells or their progeny), and then injecting the cells (or their offspring) into the targeted tissue or by systemic injection. 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 can be obtained by RNA electroporation.

[0229] The c-Kit variants and / or antigen-recognition receptors disclosed herein can be delivered to cells or subjects using any of the targeted genome editing methods. In certain embodiments, a CRISPR system is used to deliver the c-Kit variants and / or antigen-recognition receptors disclosed herein. In certain embodiments, a zinc finger nuclease is used to deliver the c-Kit variants and / or antigen-recognition receptors disclosed herein. In certain embodiments, a TALEN system is used to deliver the c-Kit variants and / or antigen-recognition receptors disclosed herein.

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

[0231] Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by combining a zinc finger DNA-binding domain with a DNA-cleaving domain. The zinc finger domain may be engineered to target specific DNA sequences, thereby enabling the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of individual ZFNs typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is by combining known, less specific zinc finger "modules." The most common cleaving domain in ZFNs is the nonspecific cleaving domain derived from the type II restriction endonuclease FokI. Using homologous DNA templates containing endogenous homologous recombination (HR) mechanisms and CAR expression cassettes, ZFNs can be used to insert CAR expression cassettes into the genome. When a target sequence is cleaved by a ZFN, the HR mechanism searches for homology between the damaged chromosome and the homologous DNA template, and then copies the template sequence between the two disrupted ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0232] Transcriptional activator-like effector nucleases (TALENs) are restriction enzymes that can be manipulated to cleave specific sequences of DNA. The TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcriptional activator-like effector DNA-binding domain with a DNA-cleaving domain. A transcriptional activator-like effector (TALE) consists of a 33-34 amino acid repeat motif with two variable positions that have strong recognition of specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be manipulated to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location within the genome. cDNA expression for use in polynucleotide therapeutics can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), Simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, nucleic acid expression can be directed using enhancers known to preferentially direct gene expression in specific cell types, if desired. Enhancers used may include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, when a genomic clone is used as a therapeutic construct, regulation may be mediated by a congeneral regulatory sequence containing any of the promoters or regulatory elements described above, or, if desired, by a regulatory sequence derived from a heterologous source.

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

[0234] 5.7. Polypeptides and Analogues The subject matter of this disclosure also includes mesothelin, CD28, CD8, CD3ζ, and c-Kit polypeptides or fragments thereof that are modified in a manner that enhances their antineoplastic activity when expressed in immune-responsive cells. The subject matter of this disclosure provides methods for optimizing amino acid sequences or nucleic acid sequences by causing changes in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of this disclosure further includes analogs of any naturally occurring polypeptides disclosed herein. Analogs may differ from naturally occurring polypeptides disclosed herein (including, but not limited to, mesothelin, CD28, CD8, CD3ζ, and c-Kit) by differences in amino acid sequences, by post-translational modifications, or both. The analog may demonstrate homology to all or part of the naturally occurring amino acid sequences of the subject matter of this disclosure to 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 higher. The length of the sequence comparison is at least 5, 10, 15 or 20 amino acid residues, e.g., at least 25, 50 or 75 amino acid residues, or more than 100 amino acid residues. Again, an exemplary approach to determining the degree of identity can be to use the BLAST program, e -3 and e -100The probability scores between these sequences indicate closely related sequences. Modifications include chemical derivatization of polypeptides in vivo and in vitro, e.g., acetylation, carboxylation, phosphorylation, or glycosylation, which can occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogues can also differ from naturally occurring polypeptides by alterations of the primary sequence. These include both natural and induced genetic variants (e.g., resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate, or by site-directed mutagenesis as described above in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al.). Cyclopeptides, molecules, and analogues containing residues other than L-amino acids, such as D-amino acids or naturally occurring or synthetic amino acids (e.g., β or γ amino acids), are also included.

[0235] In addition to full-length polypeptides, the subject of this 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 consecutive amino acids, at least 30 consecutive amino acids, or at least 50 consecutive amino acids. In certain embodiments, a fragment comprises at least 60–80, 100, 200, 300, or more consecutive amino acids. Fragments may be generated by methods known to those skilled in the art, or obtained from conventional protein processing (e.g., removal of amino acids not required for biological activity from a nascent polypeptide, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

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

[0237] 5.8. Administration The subject matter of this disclosure also provides compositions comprising the cells of this disclosure.

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

[0239] The cells of this disclosure can be administered, typically intravascularly, in any physiologically acceptable vehicle, but they can also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus). Typically, at least about 1 × 10⁻⁶ cells are present. 5 Individual cells are administered, and ultimately approximately 1 × 10⁶ cells are produced. 10 The number of cells may reach one or more. The cells of this disclosure may include a purified population of cells. Those skilled in the art can easily determine the percentage of cells of this disclosure in a population using various well-known methods, such as fluorescent cell sorting (FACS). Preferred purity ranges in a population containing the immune-responsive cells of this 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 those skilled in the art (for example, a decrease in purity may require an increase in the dosage). The cells can be introduced by injection, catheter, etc.

[0240] The compositions of the Disclosure may be pharmaceutical compositions comprising the cells of the Disclosure and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, cells may be obtained from one subject and administered to the same or different, compatible subjects. Peripheral blood-derived cells or their progeny (e.g., in vivo, ex vivo, or in vitro-derived) may be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection including parenteral administration. When the compositions of the Disclosure (e.g., pharmaceutical compositions comprising the cells of the Disclosure) are administered, they may be formulated into injectable unit dosage forms (liquids, suspensions, emulsions).

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

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

[0243] Sterile injectable solutions can be prepared by incorporating genetically modified cells, along with various amounts of other components as desired, into the required amount of a suitable solvent. Such compositions may be mixed with suitable carriers, diluents, or excipients, such as sterile water, physiological saline, glucose, or dextrose. The compositions may be lyophilized. The compositions may contain auxiliary substances, such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, colorants, etc., depending on the desired administration route and preparation. To prepare suitable preparations without unnecessary experiments, one may refer to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference.

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

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

[0246] The viscosity of the composition can be maintained at a selected level, if desired, using a pharmaceutically acceptable thickener. For example, methylcellulose is readily and economically available and readily acts with other thickeners. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The important point is to use an amount that achieves the desired viscosity. Clearly, the selection of a suitable carrier and other additives depends on the precise route of administration and the specific dosage form, e.g., the properties of the liquid dosage form (e.g., whether the composition should be formulated as a liquid, suspension, gel, or another liquid form, e.g., a time-release form or a liquid-filled form).

[0247] The amount of cells administered varies depending on the target being treated. In one embodiment, about 10 of the immune-responsive cells of this disclosure are administered. 4 ~about 10 10 Between individuals, approximately 10 4 ~about 10 6 Between individuals, approximately 10 5 ~about 10 9 Between individuals, or about 10 6 ~about 10 8 The number of cells administered to human subjects is between 1 and 10. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1 × 10 of the cells of this disclosure are administered. 5 , about 2×10 5 , about 3×10 5 , about 4×10 5 , or approximately 5 x 10 5 Individual doses are administered to human subjects. The precise determination of what is considered an effective dose may be based on factors specific to each subject, including their size, age, sex, weight, and the condition of the particular subject. The dosage can be readily determined by those skilled in the art from this disclosure and knowledge in the art.

[0248] Those skilled in the art can easily determine the amounts of cells and, as necessary, additives, vehicles, and / or carriers in the composition and to be administered in the method. Typically, any additives (in addition to the active cells and / or agents) are present in amounts of 0.001 to 50% (by weight) solution in phosphate-buffered saline, and the active ingredients are present in amounts on the order of micrograms to milligrams, for example, about 0.0001 to about 5 wt%, about 0.0001 to about 1 wt%, about 0.0001 to about 0.05 wt%, or about 0.001 to about 20 wt%, about 0.01 to about 10 wt%, or about 0.05 to about 5 wt%. With respect to any composition to be administered to animals or humans, the following can be determined: toxicity, such as determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice; the dosage of the composition(s) that elicits a suitable response, the concentration of its components, and the timing of administration of the composition(s). Such determinations can be made without unnecessary experiments, based on the knowledge of those skilled in the art, this disclosure, and the documents referenced herein. Furthermore, the timing for sequential administration can be determined without unnecessary experiments.

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

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

[0251] In certain embodiments, cells of a neoplasm or tumor have low levels of mesothelin expression (referred to as “low-MSLN-expressing cells”). In certain embodiments, low-MSLN-expressing cells are cells that express mesothelin at an expression level of about 1 / 50 or less, about 1 / 40 or less, about 1 / 30 or less, about 1 / 20 or less, about 1 / 10 or less, about 1 / 5 or less, about 1 / 4 or less, about 1 / 3 or less, or about 1 / 2 or less compared to the mesothelin expression level of normal cells.

[0252] In a particular embodiment, the solid tumor is lung cancer.

[0253] In certain embodiments, the solid tumor is mesothelioma. In certain embodiments, the mesothelioma cells are high-MSLN-expressing cells. In certain embodiments, the cells used in the treatment of high-MSLN-expressing mesothelioma cells include CARs that do not contain a costimulatory signaling region (e.g., first-generation CARs).

[0254] The subject matter of this disclosure provides methods for inducing and / or increasing an immune response in subjects in need. The cells and compositions comprising the same of this disclosure can be used in therapeutic or pharmaceutical applications. The cells and compositions comprising the same of this disclosure can be used to treat and / or prevent neoplasms in subjects. The cells and compositions comprising the same of this disclosure can be used to extend the survival time of subjects suffering from neoplasms. The cells and compositions comprising the same of this disclosure can also be used to treat and / or prevent pathogen infections or other infectious diseases in subjects, for example, immunocompromised human subjects. Such methods include the step of administering cells or compositions comprising the same (e.g., pharmaceutical compositions) to achieve a desired effect (even if it is the alleviation of an existing condition or the prevention of recurrence). In the treatment, the amount administered is an effective amount to produce the desired effect. The effective amount can be given in a single dose or in a series of doses. The effective amount can be given by a bolus or by continuous perfusion.

[0255] An effective dose (i.e., a therapeutic effective dose) is the amount sufficient to produce a beneficial or desired clinical outcome during treatment. An effective dose may be administered to a subject in one or more doses. With respect to treatment, an effective dose is sufficient to mitigate, alleviate, stabilize, reverse, or delay the progression of the disease, or otherwise reduce the pathological outcomes of the disease. The effective dose is generally determined by a physician on an individual basis and is within the scope of the skill of those skilled in the art. Several factors are typically considered when determining the appropriate dosage to achieve an effective dose. These factors include the subject's age, sex, and weight, the condition being treated, the severity of the condition, and the morphology and effective concentration of the cells being administered.

[0256] In adoptive immunotherapy using antigen-specific T cells, approximately 10 6 ~10 10 (For example, about 10 9A cell dose in the range of ) cells is typically injected. Upon administration of the cells of this disclosure to a host, and during subsequent differentiation, T cells that are specifically directed to specific antigens are induced. The modified cells may be administered by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intralymph node administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, immune-responsive cells and compositions comprising them are administered pleurally to a subject requiring them. The subject matter of this disclosure provides various methods of using cells (e.g., T cells) or compositions comprising them. For example, the subject matter of this disclosure provides a method for reducing tumor burden in a subject. In certain embodiments, the method for reducing tumor burden includes the step of administering the cells of this disclosure or compositions comprising them to a subject. The cells of this disclosure can reduce the number of tumor cells, decrease tumor size, and / or eradicate tumors in a subject. Tumors may be solid tumors. Non-exclusive examples of solid tumors include mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, and bile duct cancer.

[0257] The subject matter of this disclosure also provides methods for increasing or extending the survival time of subjects having neoplasms. In certain embodiments, the method for increasing or extending the survival time of subjects having neoplasms includes the step of administering an effective amount of the immune-responsive cells of this disclosure or a composition comprising them to the subject. The method can reduce or eradicate the tumor burden in the subject. Furthermore, the subject matter of this disclosure provides a method for increasing the immune response in a subject, which includes the step of administering the cells of this disclosure or a composition comprising them to the subject. The subject matter of this disclosure further provides a method for treating and / or preventing neoplasms in a subject, which includes the step of administering the cells of this disclosure or a composition comprising them to the subject.

[0258] As used herein, the term “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. Neoplasm growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell division and proliferation. Neoplasms can affect various cell types, tissues, or organs, including but not limited to organs selected from the group consisting of the bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nerve tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureters, urethra, uterus, and vagina, or their tissues or cell types. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In one embodiment, a neoplasm is a solid tumor. A neoplasm may be a primary tumor or primary cancer. Furthermore, the new organism may be in a state of translocation.

[0259] Cancers whose growth can be inhibited using the immune-responsive cells of the subject of this disclosure typically include cancers responsive to immunotherapy. Non-limiting examples of cancers for treatment include mesothelioma, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (e.g., metastatic breast cancer, metastatic triple-negative breast cancer), colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, cholangiocarcinoma, cervical cancer, and salivary gland cancer. Furthermore, the subject of this disclosure includes resistant or recurrent malignancies whose growth can be inhibited using the immune-responsive cells of the subject of this disclosure.

[0260] Other neoplasms or cancers that can be treated using the methods of the subject matter of this disclosure include bone cancer, intestinal cancer, liver cancer, skin cancer, head or neck cancer, melanoma (cutaneous or intraocular malignant melanoma), kidney cancer (e.g., clear cell carcinoma), pharyngeal cancer, prostate cancer (e.g., hormone-resistant prostate adenocarcinoma), hematological cancer (e.g., leukemia, lymphoma and myeloma), uterine cancer, rectal cancer, anal cancer, bladder cancer, brain cancer, stomach cancer, testicular cancer, fallopian tube cancer. Cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer This includes soft tissue sarcomas, urethral cancers, penile cancers, childhood solid tumors, lymphocytic lymphomas, bladder cancers, kidney or ureteral cancers, renal pelvis cancers, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers including those induced by asbestos, Waldenström macroglobulinemia, heavy chain disease, and Furthermore, solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, cholangiocarcinoma (nile This includes duct carcinoma, choriocarcinoma, seminomas, embryonal carcinoma, Wilms' tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrocyte, Schwann cell tumor, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0261] Patients may have an advanced form of the disease, in which case the goal of treatment may include reducing or reversing disease progression and / or alleviating side effects. Patients may have a history of a previously treated condition, in which case the goal of treatment typically includes reducing or delaying the risk of recurrence.

[0262] Human subjects suitable for treatment typically include two treatment groups that can be identified by clinical criteria. Subjects with “advanced disease” or “high tumor burden” are those with clinically measurable tumors. Clinically measurable tumors are those that can be detected based on tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; their own positive biochemical or histopathological markers are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to induce an antitumor response with the aim of alleviating these conditions. Ideally, a reduction in tumor load results, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduced risk or rate of progression, or a reduction in the pathological outcomes of the tumor.

[0263] The second group of preferred subjects is known in the art as the “adjuvant group.” These are individuals with a history of neoplasms but who have responded to other treatments. Previous treatments may include, but are not limited to, surgical resection, radiotherapy, and traditional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk of disease progression, either near the original tumor site or due to metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on features observed before and after initial treatment. These features are known in the clinical art and are suitably defined for different neoplasms. Typical features of the high-risk subgroup include tumors that invade adjacent tissues or show lymph node involvement.

[0264] Another group has a genetic predisposition to neoplasms but has not yet demonstrated the clinical signs of a neoplasm. For example, a woman who tests positive for a gene mutation associated with breast cancer but is still of childbearing age may wish to receive one or more of the immune-responsive cells described in this invention as a preventive measure to prevent the appearance of a neoplasm until she is suitable to undergo preventive surgery.

[0265] As a result of expressing c-Kit variants that enhance the antitumor effect of antigen-recognizing receptors and immune-responsive cells that bind to tumor antigens, adoptively transferred T or NK cells are given increased selective cytolytic activity at tumor sites. Furthermore, following their localization to tumors or viral infections and their proliferation, T cells transform tumor or viral infection sites into highly conductive environments for a wide range of immune cells involved in physiological antitumor or antiviral responses (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages).

[0266] Furthermore, the subject matter of this disclosure provides methods for treating and / or preventing pathogenic infections (e.g., viral, bacterial, fungal, parasitic, or protist infections) in subjects, such as those with immunocompromised conditions. The methods may include the step of administering an effective amount of the cells of this disclosure or a composition containing them to a subject having a pathogenic infection. Exemplary viral infections susceptible to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

[0267] Further modifications can be introduced into immune-responsive cells (e.g., T cells) of the present disclosure to avoid or minimize the risk of immunological complications (known as “malignant T cell transformation”), such as graft-versus-host disease (GvHD), or the risk of similar outcomes to GvHD if healthy tissue expresses the same target antigen as tumor cells. A potential solution to this problem is to manipulate suicide genes into immune-responsive 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 the EGFRt polypeptide. The EGFRt polypeptide may enable T cell elimination by administration of an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt may be covalently conjugated upstream of the antigen-recognition receptor of the CAR of the present disclosure. The suicide gene may be contained within a vector containing the nucleic acid encoding the CAR of this disclosure. In this method, administration of a prodrug designed to activate the suicide gene during malignant T cell transformation (e.g., GVHD) (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9)) induces apoptosis in suicide gene-activated CAR-expressing T cells. Incorporation of the suicide gene into the CAR of this disclosure provides an additional level of safety, along with the ability to eliminate the majority of CAR T cells within a very short period of time. Immune-responsive cells (e.g., T cells) of this disclosure into which the suicide gene has been incorporated can be preemptively eliminated at a given time after CAR T cell injection or eradicated at the earliest sign of toxicity.

[0268] Furthermore, the subject matter of this disclosure provides a method for preventing and / or treating an inflammatory disease in a subject. In certain embodiments, the method includes the step of administering the cells of this disclosure or a composition comprising them to a subject. In certain embodiments, the cells are immunosuppressive cells. In certain embodiments, the immunosuppressive cells are regulatory T cells. In one embodiment, the inflammatory disease is pancreatitis. In certain embodiments, the subject is a human. In certain embodiments, the subject is an organ transplant recipient, for example, a pancreas transplant recipient.

[0269] Furthermore, the subject matter of this disclosure provides a method for preventing graft rejection in a subject who is an organ transplant recipient. In certain embodiments, the method includes the step of administering the cells of this disclosure or a composition comprising them to the subject. In certain embodiments, the cells are immunosuppressive cells. In certain embodiments, the immunosuppressive cells are regulatory T cells. In certain embodiments, the subject is a human. In further embodiments, the subject is a pancreas transplant recipient.

[0270] In certain embodiments, the method disclosed herein further includes the step of administering a c-Kit inhibitor to a subject (e.g., as disclosed in item 5.8). The c-Kit inhibitor can inhibit the activity of c-Kit (e.g., kinase activity). In certain embodiments, c-Kit specifically inhibits a c-Kit variant, e.g., c-Kit D816V. In certain embodiments, the c-Kit inhibitor is a multityrosine kinase inhibitor. Non-limiting examples of c-Kit inhibitors include dasatinib, midostaurin, ponatinib, and imatinib. 5.11. Kit

[0271] The subject matter of this disclosure provides kits for inducing and / or enhancing an immune response in a subject, and / or treating and / or preventing neoplasms, pathogen infections, or immunodeficiencies. In certain embodiments, the kit comprises cells of this disclosure or compositions comprising them. In certain embodiments, the kit comprises a sterile container; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made from plastic, glass, laminated paper, metallic foil, or other materials suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit comprises isolated nucleic acid molecules encoding an antigen-recognition receptor (e.g., CAR or TCR) for an antigen of interest and isolated nucleic acid molecules encoding c-Kit variants in an expressible form (these may, as necessary, be contained in the same or different vectors).

[0272] If desired, cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to subjects with or at risk of developing neoplasms or pathogenic immunodeficiency. The instructions generally include information about the use of the composition for the treatment or prevention of neoplasms or pathogenic infections. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosing schedule and administration for the treatment or prevention of neoplasms, pathogenic infections, or immunodeficiency or its symptoms; precautions for use; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if any), as a label affixed to the container, or printed inside the container or on a separate sheet, brochure, card, or folder supplied with the container. [Examples]

[0273] 6. Examples The practices described herein utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are well within the scope of the art unless otherwise specified. Such techniques are well described in literature such as “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); and “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides disclosed herein and can therefore be considered when constructing and practicing the subject matter of this disclosure. Particularly useful techniques relating to specific embodiments will be discussed in subsequent sections.

[0274] The following examples are provided to give a complete disclosure and explanation of methods for preparing and using the cells and compositions of this disclosure to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention.

[0275] (Example 1) Generating structures Two constructs of the subject matter of this disclosure were generated. One construct is referred to as “M28z-KITv” (also referred to as “M28z-KITm”). The structure of M28z-KITv is shown in Figure 1A. As shown in Figure 1A, M28z-KITv comprises a c-Kit variant (e.g., c-Kit D816V) and a second-generation CAR comprising an anti-MSKN scFv, a transmembrane domain containing the CD28 polypeptide (e.g., the CD28 transmembrane domain or a portion thereof), and an intracellular domain containing CD3ζ and the CD28 polypeptide (e.g., the intracellular domain of CD28). The other construct is referred to as “Mz-KITv”. The structure of Mz-KITv is shown in Figure 1B. As shown in Figure 1B, Mz-KITv comprises a c-Kit variant (e.g., c-Kit D816V) and a first-generation CAR comprising an anti-MSKN scFv, a transmembrane domain containing the CD28 polypeptide (e.g., the CD28 transmembrane domain or a portion thereof), and an intracellular domain containing CD3ζ.

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

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

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

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

[0280] Therefore, the cKIT co-stimulatory construct demonstrated successful transduction, antigen-specific activation, and proliferation.

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

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

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

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

[0285] (Example 5) In vivo activity of the structures disclosed herein Low-mesothelin-expressing lung cancer cells (A549G) and high-mesothelin-expressing lung cancer cells (A549GM) were used to establish lung tumors in NSG mice (Figure 14C). Mice with the established low-MSLN A549G lung tumors were divided into 1 × 10⁶ mice. 5NSG mice were treated with single doses of M28z, M28z-KITv, and Mz-KITv CAR T cells. Tumor burden in NSG mice was monitored using in vivo BLI. The results are shown in Figures 12A–12D. As shown in Figures 12A–12D, when mice with tumor burden arising from cancer cells with low antigen (mesothelin) expression were treated, mice treated with M28z-KITv CAR T cells showed better and longer-lasting tumor regression compared to mice treated with M28z CAR T cells, Mz-KITv CAR T cells, or untransduced T cells (UT). Mouse survival data are shown as Kaplan-Meier survival curves in Figure 14A. As shown in Figure 14A, when mice with low antigen (mesothelin)-expressing tumors were treated, the best survival was achieved in mice treated with a single dose of M28z-KITv CAR T cells. For example, mice treated with M28z-KITv CAR T cells showed extended survival time compared to mice treated with M28z CAR T cells.

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

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

[0288] (Example 7) In vivo activity of the structures disclosed herein Lung tumors were established in NSG mice using A549GM tumor cells with high levels of mesothelin (MSLN) expression or A549G tumor cells with low levels of mesothelin (MSLN) expression. The mice were then subjected to 1 × 10⁶ 5 Patients were treated with a single dose of M28z, M28z-KITv, or Mz-KITv CAR T cells. Both Mz-KITv and M28z-KITv CAR T cells exhibited higher antitumor efficacy against high-mesothelin-expressing lung cancer cells than M28z CAR T cells (Figures 16A-16C). In low-MSLN A549G lung cancer, M28z-KITv CAR T cells showed enhanced antitumor activity compared to M28z CAR T cells, but Mz-KITv CAR T cells showed lower antitumor activity than M28z CAR T cells (Figures 17A-17C).

[0289] MSTO cells were overexpressed with low and high levels of MSLN protein to generate MG-LM cells and MGM cells, respectively (Figure 19A). In the pleural mesothelioma tumor model, mesothelioma was established in NSG mice via pleural injection of high-MSLN-expressing mesothelioma (MGM) tumor cells or low-MSLN-expressing mesothelioma (MG-LM) tumor cells. These mice were then injected with 5 × 10⁶ cells. 4 Single doses of P28z, Mz, M28z, M28z-KITv, or Mz-KITv CAR T cells were administered intrapleurally. In high-MSLN-expressing mesothelioma, Mz-KITv CAR T cells exhibited increased antitumor activity compared to Mz CAR T cells, but no significant difference was detected between M28z-KITv CAR T cells and M28z CAR T cells (Figures 18A and 18B). CAR T cell doses were intentionally reduced to mimic the low E:T ratio observed in clinics. The reduced cKIT CAR T cell dose yielded comparable antitumor efficacy compared to CD28 CAR T cells. This may be due to PDL1 / PD1 pathway-related depletion at very low E:T ratios. M28z-KITv CAR T cells also exhibited enhanced antitumor activity against low-MSLN-expressing mesothelioma (Figure 19B).

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

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

[0292] Subjective embodiments of this disclosure From the foregoing description, it is clear that changes and modifications may be made to the subject matter of this disclosure in order to adopt it for various uses and conditions. Such embodiments are also within the scope of the following claims.

[0293] In this specification, any description of an enumeration of elements in any definition of a variable includes the definition of that variable as any single element or as a combination (or subcombination) of the enumerated elements. In this specification, any description of an embodiment includes that embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0294] All patents and publications referenced in this specification are incorporated herein by reference to the same extent that each individual patent and publication is incorporated by reference specifically and individually.

Claims

1. (a) Chimeric antigen receptors (CARs) that bind to tumor antigens or pathogen antigens, and (b) Human c-Kit containing a mutation selected from D816V, D816Y, D816H, and D816F A T cell comprising a human c-Kit having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, wherein the human c-Kit results in activation of the human c-Kit independent of the interaction between the human c-Kit and its ligand.

2. The T cell according to claim 1, wherein the mutation includes or consists of D816V.

3. The T cell according to claim 1 or 2, wherein the human c-Kit contains or consists of the amino acid sequence shown in Sequence ID No. 1, except for position 816.

4. The T cell according to any one of claims 1 to 3, wherein the human c-Kit contains or consists of the amino acid sequence shown in Sequence ID No.

2.

5. The T cell according to any one of claims 1 to 4, wherein the polynucleotide encoding the human c-Kit is operably linked to an inducible promoter.

6. The T cell according to claim 5, wherein the inducible promoter is selected from activated T cell nuclear factor (NFAT), transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

7. The T cell according to any one of claims 1 to 6, wherein the CAR is recombinantly expressed.

8. The T cell according to any one of claims 1 to 7, wherein the CAR is expressed from a vector.

9. The T cell according to any one of claims 1 to 8, wherein the human c-Kit is expressed from a vector.

10. The T cell according to any one of claims 1 to 9, wherein the T cell is a cytotoxic T lymphocyte (CTL), a γδ T cell, a tumor-infiltrating lymphocyte (TIL), a regulatory T cell, or a natural killer T (NKT) cell.

11. The tumor antigens include mesothelin, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule. T cells according to any one of claims 1 to 10, selected from the group consisting of MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, tumor embryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, HPV E7 oncoprotein, and ERBB.

12. The T cell according to claim 11, wherein the tumor antigen is mesothelin.

13. The T cell according to any one of claims 1 to 12, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

14. The T cell according to claim 13, wherein the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region.

15. The T cell according to claim 14, wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide.

16. The T cell according to claim 13, wherein the CAR does not include a co-stimulatory signaling region.

17. An in vitro or ex vivo method for generating antigen-specific immune-responsive cells, comprising the steps of introducing into cells (a) a chimeric antigen receptor (CAR) that binds to a tumor antigen or pathogen antigen; and (b) a human c-Kit comprising an activating mutation selected from D816V, D816Y, D816H and D816F, wherein the human c-Kit comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, the human c-Kit results in activation of the human c-Kit independent of interaction between the human c-Kit and its ligand, and the antigen-specific immune-responsive cells are T cells.

18. A nucleic acid composition for introduction into T cells, comprising (a) a first polynucleotide encoding a chimeric antigen receptor (CAR) that binds to a tumor antigen or pathogen antigen, and (b) a second polynucleotide encoding a human c-Kit comprising an activating mutation selected from D816V, D816Y, D816H, and D816F, wherein the human c-Kit comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, and the human c-Kit results in activation of the human c-Kit independent of interaction between the human c-Kit and its ligand.

19. The nucleic acid composition according to claim 18, further comprising a first promoter operably connected to the human c-Kit.

20. The nucleic acid composition according to claim 18 or 19, further comprising a second promoter operably connected to the CAR.

21. The nucleic acid composition according to claim 19 or 20, wherein one or both of the first and second promoters are inducible promoters.

22. The nucleic acid composition according to claim 21, wherein the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter.

23. The nucleic acid composition according to any one of claims 18 to 22, wherein one or both of the first and second polynucleotides are contained in the vector.

24. The nucleic acid composition according to claim 23, wherein the vector is a retroviral vector.

25. A T cell comprising the nucleic acid composition according to any one of claims 18 to 24.

26. A pharmaceutical composition comprising T cells according to any one of claims 1 to 16 and 25, and a pharmaceutically acceptable excipient.

27. The pharmaceutical composition according to claim 26, further comprising a c-Kit inhibitor.

28. The pharmaceutical composition according to claim 27, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof.

29. A pharmaceutical composition according to any one of claims 26 to 28 for treating and / or preventing neoplasms, pathogen infections, or infectious diseases.

30. A composition comprising T cells according to any one of claims 1 to 16 and 25, or a pharmaceutical composition according to any one of claims 26 to 29, for use in a method of reducing tumor burden in a subject, wherein the method comprises the step of administering the composition or the pharmaceutical composition to the subject.

31. The composition or pharmaceutical composition according to claim 30, wherein the method reduces the number of tumor cells, reduces the size of the tumor, and / or eradicates the tumor in the subject.

32. A composition comprising T cells according to any one of claims 1 to 16 and 25, or a pharmaceutical composition according to any one of claims 26 to 29, for use in a method of treating and / or preventing neoplasms in a subject, wherein the method comprises the step of administering the composition or the pharmaceutical composition to the subject.

33. A composition comprising T cells according to any one of claims 1 to 16 and 25, or a pharmaceutical composition according to any one of claims 26 to 29, for use in a method for extending the survival time of a subject having a neoplasm, wherein the method comprises the step of administering the composition or the pharmaceutical composition to the subject.

34. The composition or pharmaceutical composition according to any one of claims 30 to 33, wherein the tumor or neoplasm is a solid tumor.

35. The composition or pharmaceutical composition according to claim 34, wherein the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, bile duct cancer, and combinations thereof.

36. The composition or pharmaceutical composition according to claim 34 or 35, wherein the solid tumor is mesothelioma.

37. The composition or pharmaceutical composition according to claim 34 or 35, wherein the solid tumor is lung cancer.

38. The composition or pharmaceutical composition according to any one of claims 30 to 37, further comprising the step of administering a c-Kit inhibitor.

39. The composition or pharmaceutical composition according to claim 38, wherein the inhibitor of c-Kit is selected from dasatinib (BMS-354825), midostaurin (PKC412), ponatinib, imatinib, and combinations thereof.

40. A kit comprising T cells according to any one of claims 1 to 16 and 25, a composition according to any one of claims 26 to 29, or a nucleic acid composition according to any one of claims 18 to 24.

41. The kit according to claim 40, further comprising written instructions for treating and / or preventing neoplasms, pathogen infections, or infectious diseases.

Images