Hypoimmunogenic modified gamma delta t cells

Abstract

The present disclosure relates to modified γδ T cells, which have reduced in vivo immunogenicity, and the use thereof.

Description

HYPOIMMUNOGENIC MODIFIED GAMMA DELTA T CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 63 / 699,017, filed on September 25, 2024, which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The content of the electronically submitted sequence listing in ASCII text file (Name: 3817_233PC01_SequenceListing_ST26.xml; Size: 10,960 bytes; and Date of Creation: September 25, 2025), filed with the application, is incorporated herein by reference in its entirety.FIELD

[0003] The present disclosure relates to modified cells, e.g., chimeric antigen receptor (CAR) and / or exogenous T cell receptor (eTCR), gamma delta (y5) T cells that have reduced in vivo immunogenicity, and the uses thereof.BACKGROUND

[0004] Cell based immunotherapy is a rapidly developing field of research for the development of novel and improved methods of treating various diseases, including cancer. Various therapies have been approved comprising isolating T cells from a subject, modifying the T cells to express a chimeric antigen receptor (CAR) or an exogenous T cell receptor (eTCR) that is capable of targeting the T cell to a particular antigen, and administering the modified T cells back to the same subject. One population of T cells of particular interest includes y5 T cells. Though less is known about the immunogenicity of y5 T cells than alpha beta T cells, reducing any potential immune response against modified y5 T cells would improve the patient experience.BRIEF SUMMARY

[0005] Some aspects of the present disclosure are directed to a modified gamma delta (y5) T cell, comprising a mutation in, or a deletion of, one or more endogenous genes encoding beta-2-microglobulin (B2M), wherein the cell has reduced or no expression of B2M protein relative to an unmodified y5 T cell.

[0006] In some aspects, the y5 T cell expresses class II major histocompatibility complex transactivator (CIITA). In some aspects, the y5 T cell expresses CD58.

[0007] Some aspects of the present disclosure are directed to a modified y5 T cell, comprising a mutation in, or a deletion of, one or more endogenous genes encoding CIITA, wherein the cell has reduced or no expression of CIITA protein relative to an unmodified y5 T cell. In some aspects, the y5 T cell expresses B2M. In some aspects, the y5 T cell expresses CD58.

[0008] Some aspects of the present disclosure are directed to a modified y5 T cell, comprising a mutation in, or a deletion of, one or more endogenous genes encoding CD58, wherein the cell has reduced or no expression of CD58 protein relative to an unmodified y5 T cell. In some aspects, the y5 T cell expresses B2M. In some aspects, the y5 T cell expresses CIITA.

[0009] In some aspects, the modified y5 T cell further comprises an increased expression of HLA-E relative to an unmodified y5 T cell. In some aspects, the increased expression of the HLA-E results from: (i) a transfection of a nucleic acid encoding an HLA-E polypeptide; (ii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii).

[0010] In some aspects, the mutation in, or a deletion of, the one or more endogenous genes comprises a mutation in the one or more endogenous genes that results in a loss of expression of a protein encoded by the endogenous gene. In some aspects, the mutation in, or a deletion of, the one or more endogenous genes comprises a deletion in the one or more endogenous genes that results in a loss of expression of a protein encoded by the endogenous gene.

[0011] In some aspects, the modified y5 T cell is a V51+cell. In some aspects, the modified y5 T cell is a V52+cell. In some aspects, the modified y5 T cell is a human y5 T cell. In some aspects, the modified y5 T cell is obtained from blood or skin.

[0012] In some aspects, the modified y5 T cell further comprises a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR). In some aspects, the CAR or the eTCR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the tumor antigen comprises CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL- HRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART- 1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE, claudin, B7H3 or any combination thereof. In some aspects, the modified y5 T cell comprises a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds mesothelin.

[0013] Some aspects of the present disclosure are directed to a method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding B2M in a y5 T cell, wherein the expression of B2M protein is reduced relative to the expression of B2M by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of CIITA and CD58.

[0014] Some aspects of the present disclosure are directed to a method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding CIITA in a y5 T cell, wherein the expression of CIITA protein is reduced relative to the expression of CIITA by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CD58.

[0015] Some aspects of the present disclosure are directed to a method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding CD58 in a y5 T cell, wherein the expression of CD58 protein is reduced relative to the expression of CD58 by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CIITA.

[0016] In some aspects, the method further comprises (i) introducing into the cell, a nucleic acid encoding an HLA-E polypeptide; (ii) modifying an endogenous gene encoding anHLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii).

[0017] In some aspects, the one or more endogenous genes are genetically modified using a gene editing tool. In some aspects, the gene editing tool comprises a CRISPR / Cas9, CRISPR / Casl2, TALEN, a zinc-finger endonuclease, or any combination thereof. In some aspects, the one or more endogenous genes are genetically modified by introducing into the cell CRISPR / Cas9 and a polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences within the endogenous gene.

[0018] In some aspects, the y5 T cell is a V51+cell. In some aspects, the y5 T cell is a V52+cell. In some aspects, the y5 T cell is a human y5 T cell. In some aspects, the y5 T cell is obtained from blood or skin.

[0019] In some aspects, the method further comprises introducing into the y5 T cell, a nucleic acid encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR). In some aspects, the CAR or the eTCR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the tumor antigen comprises CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c- Met, mesothelin, GD3, HERV-K, IL-llRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, BCMA, GCC, ADGRE, claudin or any combination thereof. In some aspects, the tumor antigen comprises mesothelin.

[0020] In some aspects, the method further comprises transfecting the y5 T cell with a nucleic acid encoding an interleukin 15 (IL15) polypeptide. In some aspects, the IL15 polypeptide is a membrane bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra).

[0021] Some aspects of the present disclosure are directed to a modified y5 T cell prepared according to a method disclosed herein. In some aspects, the modified y5 T cell has increased persistence in vivo relative to an unmodified y5 T cell.

[0022] Some aspects of the present disclosure are directed to a population of cells comprising a modified y5 T cell disclosed herein.

[0023] Some aspects of the present disclosure are directed to a cell population, wherein at least 50% of the cells in the population comprises a modified y5 T cell disclosed herein.

[0024] Some aspects of the present disclosure are directed to a method of treating a subject in need thereof, comprising administering to the subject a modified y5 T cell disclosed herein.

[0025] In some aspects, the subject is afflicted with a cancer. In some aspects, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0026] FIG. l is a graphical representation, illustrating mixed lymphocyte reaction (MLR) data with VD1 y5 T cell donors. FIG. 1 shows MLR data of PBMC of Donor 3 in response to mitomycin-treated VD1 y5 T cells. The % PBMC proliferation was measured in HLA mismatched PBMC donors and HLA mismatched / prevalence PBMC donors.

[0027] FIG. 2 is a graphical representation, illustrating mixed lymphocyte reaction (MLR) data with VD1 y5 T cell donors. FIG. 2 shows MLR data of multiple PBMC donors in response to mitomycin-treated VD1 y5 T cells. The % PBMC proliferation was measuredin HLA mismatched / prevalence PBMC donors. VD1 y5 T cells triggered lower alloreactivity as measured by PBMC proliferation in all HLA mismatched PBMC donors, compared to iPSC-derived T cells ("iT cells").

[0028] FIGs. 3 A-3B are graphical representations, illustrating % proliferated PBMCs. FIG. 3 A shows the percent of proliferated PBMCs in donor 1 y5 T and aP T cells of PBMC A, PBMC B, PBMC C, and PBMC D donors. FIG. 3B shows the percent of proliferated PBMCs in donor 3 y5 T and aP T cells of PBMC A, PBMC B, PBMC C, and PBMC D donors.

[0029] FIGs. 4A-4H are graphical representations, illustrating the fold expansion of gdT VD1 cells, comparing wild type gdT VD1 cells and gene edited gdT VD1 cells. FIG. 4A shows live cell fold expansion of wild type (wt) donor 3, B2M KO, and B2M & CIITA KO on day 4 and day 14. FIG. 4B shows y5 T cell viability of wild type (wt) donor 3, B2M KO, and B2M & CIITA KO on day 4 and day 14. FIGs. 4C-4F are heat maps illustrating proliferation of PBMC D cells where: PBMC D cells are not exposed to a second lymphocyte (FIG 4C), PBMC D is treated with PH A (FIG. 4D), PBMC D is contacted with wild type y5 T cells (FIG. 4E), PBMC D is treated with B2M KO (FIG. 4F) and B2M / CIITA dKO y5 T cells (FIG. 4G) in a MLR assay. FIG. 4C shows PBMC only. FIG. 4D shows PBMC treated with PHA which is intended to be a control stimulation of PBMC proliferation. FIG. 4E shows PBMC and wild type y5 T cells. FIG. 4F shows PBMC and B2M KO y5 T cells. FIG. 4G shows PBMC and dKO y5 T cells. FIG. 4H shows the percent of proliferation for PBMCs for PBMCs alone, PBMCs plus PHA, PBMCs plus wild type y5 T cells, PBMCs plus B2M KO y5 T cells, and PBMCs plus dKO y5 T cells.

[0030] FIGs. 5A-5B are graphical representations, illustrating y5 T cell viability and y5 T cell death by NK cells (two donors, E and F). FIGs. 5A and 5B show percent target cell death CTV+ of NK donor cells co-cultured with B2M KO, y5 T DKO, and wild type y5 T cells at 5: 1, 2: 1, 1 : 1, and no target cells E:T ratios.

[0031] FIGs. 6A-6C are graphical representations, illustrating PBMC proliferation (FIG. 6A) and y5 T cell killing by NK cells (FIGs. 6B and 6C) for wild type y5 T cells and B2M KO, B2M + HLA-E, B2M + HLA-E + FASDN, DKO, DKO + HLA-E, DKO + HLA-E FASDN, and CIITA KO y5 T cells.

[0032] FIGs. 7A-7E are graphical representations, illustrating cell expansion (FIG. 7A), cell viability (FIG. 7B), PBMC proliferation (FIG. 7C), and NK killing (FIGs. 7D-7E) for y5 T cells having a single knock out of B2M, CD58, or HLA-A.DETAILED DESCRIPTION

[0033] Some aspects of the present disclosure are directed to modified gamma delta (y5) T cells, comprising a mutation in one or more endogenous genes encoding beta-2- microglobulin (B2M) (e.g., wherein the one or more gene(s) encoding B2M are mutated and / or deleted and any gene(s) encoding CIITA and / or CD58 are not mutated and / or deleted). In some aspects, the cell has reduced or no expression of B2M protein relative to an unmodified y5 T cell. Some aspects of the present disclosure are directed to modified y5 T cells, comprising a deletion of one or more endogenous genes encoding B2M, wherein the cell has reduced or no expression of B2M protein relative to an unmodified y5 T cell. In some aspects, the modified y5 T cell does not comprise a deletion or mutation in a gene encoding CIITA or CD58.

[0034] Some aspects of the present disclosure are directed to modified y5 T cells, comprising a mutation in one or more endogenous genes encoding CIITA (e.g., wherein the one or more gene(s) encoding CIITA are mutated and / or deleted and any gene(s) encoding B2M and / or CD58 are not mutated and / or deleted). In some aspects, the cell has reduced or no expression of CIITA protein relative to an unmodified y5 T cell. Some aspects of the present disclosure are directed to modified y5 T cells, comprising a deletion of one or more endogenous genes encoding CIITA, wherein the cell has reduced or no expression of CIITA protein relative to an unmodified y5 T cell. In some aspects, the modified y5 T cell does not comprise a deletion or mutation in a gene encoding B2M or CD58.

[0035] Some aspects of the present disclosure are directed to modified y5 T cells, comprising a mutation in one or more endogenous genes encoding CD58 (e.g., wherein the one or more gene(s) encoding CD58 are mutated and / or deleted and any gene(s) encoding CIITA and / or B2M are not mutated and / or deleted). In some aspects, the cell has reduced or no expression of CD58 protein relative to an unmodified y5 T cell. Some aspects of the present disclosure are directed to modified y5 T cells, comprising a deletion of one or more endogenous genes encoding CD58, wherein the cell has reduced or no expression of CD58 protein relative to an unmodified y5 T cell. In some aspects, the modified y5 T cell does not comprise a deletion or mutation in a gene encoding CIITA or B2M.

[0036] In some aspects, the reduced expression or deletion of the endogenous gene results from a mutation in, insertion of a transgene into or a deletion (knock-out) of the endogenous gene.

[0037] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0038] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.I. Terms

[0039] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0040] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0041] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a chimeric polypeptide," is understood to represent one or more chimeric polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0042] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase suchas "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In addition, "or" is used mean an open list of the components in the list. For example, "wherein X comprises A or B" means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.

[0043] The terms "about" or "comprising essentially of' refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, ie., the limitations of the measurement system. For example, "about" or "comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of should be assumed to be within an acceptable error range for that particular value or composition.

[0044] The terms "activated T cells" and "activated y5 T cells" refer to, among other things, y5 T cells T cells that are undergoing cell division.

[0045] An "antigen" refers to any molecule, e.g., a peptide, that provokes an immune response or is capable of being bound by a TCR. The immune response may involve antibody production, the activation of specific immunologically-competent cells, or a combination thereof. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be endogenously expressed, i.e. expressed by genomic DNA, or can be recombinantly expressed. An antigen and / or an epitope can be specific to a certain tissue, such as a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one aspect, antigens are tumor antigens.

[0046] An "antigen-presenting cell" or "APC," as used herein, refers to a cell or a cell-like antigen-presenting surface that expresses one or more antigen. In some aspects, the antigen is displayed on the surface of the APC.

[0047] An "anti-tumor effect" as used herein, refers to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumorcell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival of a patient, an increase in life expectancy of a patient, or amelioration of various physiological symptoms in a patient associated with the tumor. An anti-tumor effect can also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.

[0048] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term "approximately" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0049] The term "autologous" refers to any material, e.g., a y5 T cell, derived from the same individual to which it is later to be re-introduced. For example, an autologous y5 T cell therapy comprises administering to a subject a y5 T cell that was isolated from the same subject. The term "allogeneic" refers to any material derived from one individual which is then introduced to another individual of the same species. For example, an allogeneic y5 T cell transplantation comprises administering to a subject a y5 T cell that was obtained from a donor other than the subject.

[0050] A "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A "cancer" or "cancer tissue" can include a tumor. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system including lymphoma, leukemia, and other leukocyte malignancies. In some aspects, the methods of the present disclosure can be used to reduce the tumor size of a tumor derived from, for example, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, breast cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. The particular cancer can be responsive to chemo- or radiation therapy or the cancer can be refractory. A refractory cancer refers to a cancer that is not amendable to surgical intervention, and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.

[0051] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of' are also provided.

[0052] A "cytokine," as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine can be endogenously expressed by a cell, added to a cell in culture, administered to a subject, or any combination thereof. Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-21, and interferon (IFN) gamma. Examples of pro- inflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growthfactor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0053] Chemokines" are a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL- 16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein la (MIP-la, MIP- la), MIP-Ib (MIP-lb), gamma-induced protein 10 (IP- 10), and thymus and activation regulated chemokine (TARC or CCL17).

[0054] Other examples of cytokines include, but are not limited to chemokine (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL 13, IL-1, IL-3, IL-9, IL-11, IL- 12, IL- 14, IL- 17, IL-20, IL-21, granulocyte colony- stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), CD 154, lymphotoxin (LT) beta, 4- IBB ligand (4-1BBL), a proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

[0055] The term "engineered Autologous Cell Therapy," which can be abbreviated as "eACT™," also known as adoptive cell transfer, is a process by which a patient's own immune cells, e.g., T cells and / or NK cells, are collected and subsequently genetically altered to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. Immune cells, e.g., T cells, can be engineered to express, for example, chimeric antigen receptors (CAR) or T cell receptor (TCR). CAR positive (+)T cells are engineered to express an extracellular single chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling part comprising a costimulatory domain and an activating domain. The costimulatory domain can be derived from, e.g., CD28, and the activating domain can be derived from, e.g., CD3-zeta (figure 1). In certain aspects, the CAR is designed to have two, three, four, or more costimulatory domains. The CAR scFv can be designed to target, for example, CD 19, which is a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignances, including but not limited to NHL, CLL, and non- T cell ALL. Example CAR+ T cell therapies and constructs are described in U.S. PatentPublication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, and these references are incorporated by reference in their entirety.

[0056] An "immune response" is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+cell, a CD8+T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell. In some aspects, an immune response refers to NK cell-mediated killing of a foreign cell, e.g., an allogeneic y5 T cell therapy.

[0057] "Immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0058] As used herein, the term "inactivating" or "inactivation," e.g., in reference to a gene or protein, refers to a measure that can induce the reduced expression of the protein. In some aspects, inactivation can be achieved by a deletion or mutation of all or a part of the coding region of a gene or all or part of a non-coding region of a gene that results in decreased expression of the gene or the protein encoded by the gene. In some aspects, inactivation is achieved by deletion of the entire coding region of a gene. In some aspects, inactivation is achieved by partial deletion of a coding region of a gene. In some aspects, inactivation is achieved by deletion of one or more regulatory elements that facilitate expression of the gene. In some aspects, inactivation is achieved by a mutation in one or more regulatory elements that results in decreased expression or loss of expression of the gene. In some aspects, inactivation is achieved by mutation of one or more nucleic acid that results in the expression of a non-functional protein. In some aspects, inactivation is achieved by a missense mutation that results in the expression of a non-functional protein.In some aspects, inactivation is achieved by interference of the transcription or translation of a gene that results in the reduced expression of the protein. In some aspects, decreased expression is relative to the expression of the target gene in the cell prior to modification (e.g., deletion or mutation). In some aspects, the expression of the gene is measured prior to modification, then the cell is modified, and then the expression of the gene is measured following modification.

[0059] As used herein, the term "introducing" or "introduction" refers to expressing a heterologous polynucleotide and / or polypeptide in a cell. In some aspects, introduction is achieved by transfecting the cell with a polynucleotide of interest. In some aspects, introduction is achieved by genetically modifying the cell to express a heterologous sequence, e.g., using a gene editing tool including, but not limited to, CRISPR / Cas, CRISPR / Cas9, CRISPR / Cas 12, CRISPR / Cas 12a, CRISPR / Cpfl, zinc finger, TALEN, Closver-Cas or a variant thereof. In some aspects, introduction is achieved by contacting the cell with an mRNA encoding a polypeptide of interest, such that the mRNA enters into the cell or the nucleus of the cell. In some aspects, introduction comprises transfecting or transducing a cell with a polynucleotide encododing a popypeptide.

[0060] As used herein, the term "iPS cell" or "iPSC" refers to a cell that has been dedifferentiated (or reprogrammed) into a more naive, e.g., pluripotent, state. Various methods of de-differentiating a cell are known, including, but not limited to, overexpressing Oct3 / 4, Sox2, Klf4, and c-Myc (the '"Yamanaka factors") in the cell (see, e.g., Takahashi and Yamanaka, Cell 126.663-r16 (2006)). In some aspects, the iPS cell is a pluripotent cell, e.g, capable of differentiation into a limited number of cell types. In some aspects, the iPS cell is a totipotent cell, e.g, capable of differentiation into any cell type. In some aspects, an iPS cell can be re- differentiated into a specific type of cell, e.g., an immune cell, e.g., a y5 T cell. In some aspects, a T cell is derived from an iPSC to generate an induced T cell or "iT cell."

[0061] The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells (alpha beta T cells and y5 T cells), or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the inherent immune system. NK cells reject tumors and cells infected by viruses by inducing apoptosis or programmed cell death in the target cell. They were termed "natural killers" because NK cells do not require activation in order to kill a target cell. T-cells play a major role in cell-mediated-immunity. T-cell receptors (TCR) expressed on the surface of T cells differentiate T cells from otherlymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for T cell maturation. There are six types of T-cells, namely: Helper T-cells (e.g. CD4+ cells); Cytotoxic T-cells (also known as TC, cytotoxic T lymphocyte, CTL, T- killer cell, cytolytic T cell, CD8+ T-cells or killer T cell); Memory T-cells ((i) stem memory TSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2R.p, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not IFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNy and IL-4); Regulatory T-cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells); Natural Killer T-cells (NKT); and Gamma Delta T-cells.

[0062] B-cells play a principal role in humoral immunity (with antibody involvement). A B cell makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

[0063] As used herein, the term "gamma delta T cell" or "y5 T cell" refers to a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 T cells can be broadly classified as one of two types: peripheral blood-resident y5 T cells and tissue-resident y5 T cells. Most blood-resident y5 T cells express a V52 TCR, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 and / or other V5 chains.

[0064] As used herein, an "MHC class I molecule" refers to a protein product of a wildtype or variant HLA class I gene encoding an MHC class I molecule. Accordingly, "HLA class I molecule" and "MHC class I molecule" can be used interchangeably. The MHC Class I molecule comprises two protein chains: the alpha chain and the p2-microglobulin (P2m) chain. Human P2m is encoded by the B2M gene. The amino acid sequence of P2m is set forth in SEQ ID NO: 1 (Table 1). In some aspects, the P2m is encoded by the nucleic acid sequence set forth in SEQ ID NO: 2 (Table 2). The alpha chain of the MHC Class I molecule is encoded by the HLA gene complex. The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. The HLA gene are highly variant, with over 20,000 HLA alleles and related alleles, including over 15,000 HLA Class I alleles, known in the art, encodingthousands of HLA proteins, including over 10,000 HLA Class I proteins. There are at least three genes in the HLA complex that encode an MHC Class I alpha chain protein: HLA-A, HLA-B, and HLA-C. In addition, HLA-E, HLA-F, and HLA-G encode proteins that associate with the MHC Class I molecule.Table 1. Amino Acid Sequence of Human P2mTable 2. Nucleic Acid Sequence of Human P2m

[0065] As used herein, an "MHC class II molecule" refers to a protein product of a wildtype or variant HLA class II gene encoding an MHC class II molecule. Accordingly, "MHC class II molecule" can be used interchangeably with an "HLA class II molecule." A typical MHC Class II molecule comprises two protein chains: an alpha chain and a beta chain. In general, naturally occurring alpha chains and beta chains each comprise a transmembrane domain, which anchors the alpha / beta chain to the cell surface, and an extracellular domain, which carries the antigen and interacts with a TCR and / or CD4 expressed on a T cell. Both the MHC Class II alpha and beta chains are encoded by the HLA gene complex. The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. The HLA gene complex is highly variant, with over 20,000 HLA alleles and related alleles, including over 250 MHC class II alpha chain alleles and 5,000 MHC class II beta chain alleles, known in the art, encoding thousands of MHC class II proteins. Three loci in the HLA complex encode MHC Class II proteins: HLA-DP, HLA-DQ, and HLA-DR. HLA-DO and HLA-DM encode proteins that associate with the MHC class II molecule and support its configuration and function.

[0066] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0067] "Potentiating an endogenous immune response" means increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0068] The term "recombinant" or "modified" cell, as used herein, is intended to refer to a cell, e.g., a y5 T cell, that comprises a nucleic acid that is not naturally present in the cell, and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Though certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny are still included within the scope of the term "recombinant" or "modified" as used herein.

[0069] As used herein, the terms "reduced expression" and "increased expression" refer to the expression of a particular gene or protein in a cell relative to a control, e.g., the expression of a particular gene in a modified cell as compared to the expression of the gene in a wild-type (unmodified) cell. The relative expression can be based on mRNA levels and / or protein levels. Any means of measuring the level of mRNA and / or protein can be used to determine whether a gene or protein has reduced or increased expression, including but not limited to immunohistochemistry and PCR-based techniques.

[0070] In some aspects, a cell that has "reduced expression" of a particular gene or protein has an expression level that is at less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 90%, less than about 85%,less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% that of the expression of the gene or protein in an unmodified cell, e.g., wild-type cell of the same cell type. In some aspects, the unmodified cell, e.g., wild-type cell of the same cell type, expresses the particular gene or protein, and the modified cell has no detectable level of expression of the protein or gene, e.g., a "knock-out" of the gene or protein. As such, the term "knock-out" refers to the complete ablation of expression of a particular gene or protein, such that there is no detectable level of expression of the gene or protein in the cell.

[0071] In some aspects, a cell that has "increased expression" or “overexpression” of a particular gene or protein has an expression level that is more than about 105%, more than about 110%, more than about 115%, more than about 120%, more than about 125%, more than about 130%, more than about 140%, more than about 150%, more than about 160%, more than about 170%, more than about 180%, more than about 190% more than about 200%, more than about 225%, more than about 250%, more than about 275%, more than about 300%, more than about 350%, more than about 400%, more than about 450%, more than about 500%, more than about 600%, more than about 700%, more than about 800%, more than about 900%, or more than about 1000% that of the expression of the gene or protein in an unmodified cell, e.g., a wild-type cell of the same cell type or population of cells or the same cell or population of cells prior to the modification, wherein 100% expression corresponds to that observed in case of the wild-type cell. In some aspects, a cell that has "increased expression" or “overexpression” of a particular gene or protein has an expression level that at least about 5% higher, at least about 10% higher, at least about 15% higher, at least about 20% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least about 45% higher, at least about 50% higher, at least about 55% higher, at least about 60% higher, at least about 65% higher, at least about 70% higher, at least about 75% higher, at least about 80% higher, at least about 85% higher, at least about 90% higher, at least about 95% higher, at least about 100% higher, at least about 110% higher, at least about 120% higher, at least about 130% higher, at least about 140% higher, at least about 150% higher, at least about 160% higher, at least about 170% higher, at least about 180% higher, at least about 190% higher, at least about 200% higher, at least about 250% higher, at least about 300% higher, at least about350% higher, at least about 400% higher, at least about 450% higher, at least about 500% higher, at least about 600% higher, at least about 700% higher, at least about 800% higher, at least about 900% higher, or at least about 1000% higher than the expression of the gene or protein in an unmodified cell, e.g., a wild-type cell of the same cell type or population of cells or the same cell or population of cells prior to the modification. In some aspects, the unmodified cell, e.g., wild-type cell of the same cell type or the same cell or population of cells prior to the modification, has no expression of the particular gene or protein, and the increased expression is any expression of the gene or protein.

[0072] Increased expression of a particular gene or protein can be achieved by any method. In some aspects, the expression of a gene or polypeptide is increased by introducing into the cell, a molecule, signal, element or modification that results in increased expression of a gene or polypeptide in the cell. In some aspects, the expression of a gene or polypeptide is increased by transfecting the cell with a nucleic acid molecule encoding a protein. In some aspects, a nucleic acid of interest is introduced into the cell via eletroporation. In some aspects, the nucleic acid is a vector. In some aspects, the nucleic acid comprises an mRNA. In some aspects, the expression of a gene or protein is increased by modifying an endogenous regulatory element or inserting a heterolgous regulatory element into an endogenous gene thereby to increase expression of the endogenous gene encoding a polypeptide of interest. In some aspects, the expression of a gene or protein is increased by knocking-in a heterolgous coding region that encodes the polypeptide of interest. In some aspects, modification of an endogenous sequence is achieved using a gene editing tool, such as CRISPR.

[0073] As used herein, the terms "subject" and "patient" are used interchangeably and refer to either a human or a non-human, such as primates, mammals, and vertebrates. In particular aspects, the subject is a human.

[0074] The term "chimeric antigen receptor" or "CAR," as used herein, refers to a recombinant fusion protein that has an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain. In some aspects, a chimeric antigen receptor disclosed herein comprises a chimeric polypeptide of the present disclosure.

[0075] The term "T cell receptor" (TCR), as used herein, refers to a heteromeric cellsurface receptor capable of specifically interacting with a target antigen. As used herein, "TCR" includes but is not limited to naturally occurring and non-naturally occurring TCRs;full-length TCRs and antigen binding portions thereof; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In human, TCRs are expressed on the surface of T cells, and they are responsible for T cell recognition and targeting of antigen presenting cells. Antigen presenting cells (APCs) display fragments of foreign proteins (antigens) complexed with the major histocompatibility complex (MHC; also referred to herein as complexed with an HLA molecule, e.g., an HLA class 1 molecule). A TCR recognizes and binds to the antigen-HLA complex and recruits CD3 (expressed by T cells), activating the TCR. The activated TCR initiates downstream signaling and an immune response, including the destruction of the antigen-presenting cell.

[0076] An "exogenous TCR" or an "eTCR," as used herein, refers to a TCR that is heterologous to the cell which expresses the TCR. As used herein, the term "heterologous" refers to something that is not native to or naturally found in, e.g., the paticular cell.

[0077] In general, a TCR can comprise two chains, (i) an alpha chain and a beta chain (alpha-beta TCR) for alpha-beta T cells, or (ii) a gamma chain and a delta chain (y5 TCR) for y5 T cells, interconnected by disulfide bonds. Each chain comprises a variable domain (alpha chain variable domain, beta chain variable domain, gamma chain variable domain, and delta chain variable domain) and a constant region (alpha chain constant region, beta chain constant region, gamma chain constant region, and delta chain constant region). The variable domain is located distal to the cell membrane, and the variable domain interacts with an antigen. The constant region is located proximal to the cell membrane. A TCR can further comprises a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the transmembrane region and the cytoplasmic tail, when present, as well as the traditional "constant region."

[0078] The variable domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each alpha chain variable domain and beta chain variable domain comprises three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Each variable domain contains a binding domain that interacts with an antigen. Though all three CDRs on each chain are involved in antigen binding, CDR3 is believed to be the primary antigen binding region. CDR1 is also interacts with the antigen, while CD2 is believed to primarily recognize the ELLA complex. In one aspect, the gamma chain variable domain include V gamma 1, V gamma 2, V gamma 3, V gamma 4, V gamma 5, V gamma 6, V gamma 7, V gamma 8 and V gamma 9, and examples of delta chainvariable domain include V delta (V5) 1, V52, V53, V54, V55, V56, V57, V58 and V59. While the combination of specific gamma chain variable domain and delta chain variable domain in the TCR is not limited, for example, eTCR is any of V gamma 3-V delta 1 TCR (y351TCR), V gamma 4-V delta 1 TCR (54vlTCR), V gamma 9-V delta 1 TCR (5951 TCR) and V gamma 9-V delta 2 TCR (5952TCR).

[0079] Where not expressly stated, and unless the context indicates otherwise, the term "TCR" also includes an antigen-binding fragment or an antigen-binding portion of any TCR disclosed herein, and includes a monovalent and a divalent fragment or portion, and a single chain TCR. The term "TCR" is not limited to naturally occurring TCRs bound to the surface of a T cell. As used herein, the term "TCR" further refers to a TCR described herein that is expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or that is modified to express CD3, as described herein), or a TCR described herein that is free from a cell membrane (e.g., an isolated TCR or a soluble TCR).

[0080] A "TCR fragment," "antigen binding molecule," or "portion of a TCR" refers to any portion of a TCR less than the whole. An antigen binding molecule can include the antigenic complementarity determining regions (CDRs).

[0081] A "suicide gene" refers to a gene that causes a cell to kill itself. In some aspects, the suicide gene causes a cell to kill itself through apoptosis. Non-limiting examples of suicide genes include viral thymidine kinase, cytosine deaminases, intracellular antibody against antioxidative enzymes (AOEs), bacterial nitroreductase, caspase and DNase.

[0082] In one aspect, the suicide gene is viral thymidine kinase (TK). Thymidine kinase is an ATP -thymidine 5'-phosphotransferase that converts deoxythymidine intodeoxythymidien 5'-monophosphate, which is further phosphorylated to deoxythymidine diphosphate and thereafter to deoxy thy mi dine triphosphate by viral thymidine kinase and nucleoside diphosphate kinase respectively. Deoxythymidine triphosphate is incorporated into the synthesized DNA molecule by DNA polymerase. Some dNTP analogs, such as Ganciclovir (GCV), a synthetic analogue of 2'-deoxy- guanosine, have the ability to terminate the DNA synthesis upon their incorporation into synthesized DNA. Termination of synthesis triggers the apoptotic signaling cascades. While GCV is not recognized by human thymidine kinase, it is recognized as a substrate for some viral thymidine kinase, such as Herpes Simplex Virus- 1 thymidine kinase (HSV- TK). As a result, a human cell expressing HSV-TK converts GCV into GCV phosphate, which is further phosphorylated and incorporated into the synthesized DNA, leading to thetermination of synthesis and apoptosis. While the variant of HSV-TK is not limited, HSV- TK is, for example, TK007 (see Preuss et al., Hum Gene Ther. 2010 Aug; 21(8): 929-41).

[0083] In some aspects, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyze cytosine to uracile with release of ammonia. In physiological conditions, the modified site is recognized by endonucleases, then the phophodiester bond in the DNA is broken, initiating repair by incorporation of a new cytosine. However, cytosine deaminase can also converts 5 -fluorocytosine into 5 -fluorouracil (5-FU). Therefore, upon provision of non-toxic prodrug 5-FC, cytosine deaminase converts it into highly toxic 5-FU (a suicide inhibitor of thymidylate synthetase), leading to the inhibition of cell growth and apoptosis.

[0084] The term "therapeutic benefit" or "therapeutically effective," as used herein, refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

[0085] As used herein, the term "treating" or "treatment" of a disease or condition refers to executing a protocol, which may include administering one or more therapies to a patient, in an effort to alleviate signs or symptoms of the disease. In some aspects, a treatment decreases the rate of disease progression, ameliorates or palliates the disease state, and / or facilitates remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, in some aspects, "treating" or "treatment" includes "preventing" or "prevention" of a disease or an undesirable condition. However, "treating" or "treatment" does not require complete alleviation of all signs and / or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0086] In various aspects, a subject in need thereof is treated for a disease or for alleviating symptoms associated with a disease (e.g., a cancer) using a population of modified cells described herein. In some aspects, the modified cells are y5 T cells that are transduced with a CAR or a TCR and are further modified to exhibit reduced or no expression of B2M protein, CD58, or CIITA, such that the modified cells are less susceptible to killing by the subject’s immune system when such cells are administered to the subject.

[0087] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which thisdisclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0089] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0090] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0091] Various aspects described herein are described in further detail in the following subsections.II. Compositions of the Disclosure

[0092] Some aspects of the present disclosure are directed to modified y5 T cells, having reduced or no expression of B2M, CIITA, or CD58. The present disclosure provides that allogenic y5 T cells with lower expression of B2M, CIITA, or CD58 are more readily accepted by a host patient than un-modified y5 T cells. Patients receiving the modified y5 T cells of the present disclosure have are expected to have a reduced immune response to the administered y5 T cells than to unmodified y5 T cells, allowing for more of the transfected y5 T cells to survive after administration. Surprisingly, it was found that having a reduction of only one of B2M, CIITA, and CD58, while not modifying the expression of the other two genes, results in the highest likelihood of avoiding an immune response against the administered y5 T cells.ILA. Modified Cells

[0093] Some aspects of the present disclosure are directed to modified y5 T cells, comprising a mutation in one or more endogenous genes encoding beta-2-microglobulin (B2M). In some aspects, the cell has reduced or no expression of B2M protein relative to an unmodified y5 T cell. Some aspects of the present disclosure are directed to modified y5T cells, comprising a deletion of one or more endogenous genes encoding B2M, wherein the cell has reduced or no expression of B2M protein relative to an unmodified y5 T cell. In some aspects, the modified y5 T cells express CIITA. In some aspects, the modified y5 T cells express CD58. In some aspects, modified y5 T cells (i) have reduced expression of B2M protein relative to an unmodified y5 T cell, (ii) express CIITA, and (iii) express CD58. In some aspects, modified y5 T cells (i) have no detectable expression of B2M protein, (ii) express CIITA, and (iii) express CD58. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous CIITA gene. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous CD58 gene. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous CIITA gene or an endogenous CD58 gene. In some aspects, the modified y5 T cells do not comprise a deletion in an endogenous CIITA gene or an endogenous CD58 gene. In some aspects, the modified y5 T cells do not comprise a mutation in an endogenous CIITA gene or an endogenous CD58 gene.

[0094] In some aspects, the expression of the B2M is less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% the B2M expression in a wild-type cell of the same cell type. In some aspects, the cell has no detectable expression of the B2M.

[0095] Some aspects of the present disclosure are directed to modified y5 T cells, comprising a mutation in one or more endogenous genes encoding CIITA. In some aspects, the cell has reduced or no expression of CIITA protein relative to an unmodified y5 T cell. Some aspects of the present disclosure are directed to modified y5 T cells, comprising a deletion of one or more endogenous genes encoding CIITA, wherein the cell has reduced or no expression of CIITA protein relative to an unmodified y5 T cell. In some aspects, the modified y5 T cells express B2M. In some aspects, the modified y5 T cells express CD58. In some aspects, modified y5 T cells (i) have reduced expression of CIITA protein relative to an unmodified y5 T cell, (ii) express B2M, and (iii) express CD58. In some aspects, modified y5 T cells (i) have no detectable expression of CIITA protein, (ii) express B2M, and (iii) express CD58. In some aspects, the modified y5 T cells do not comprise a mutationor deletion in an endogenous B2M gene. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous CD58 gene. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous B2M gene or an endogenous CD58 gene. In some aspects, the modified y5 T cells do not comprise a deletion in an endogenous B2M gene or an endogenous CD58 gene. In some aspects, the modified y5 T cells do not comprise a mutation in an endogenous B2M gene or an endogenous CD58 gene.

[0096] In some aspects, the expression of the CIITA is less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% the CIITA expression in a wild-type cell of the same cell type. In some aspects, the cell has no detectable expression of the CIITA.

[0097] Some aspects of the present disclosure are directed to modified y5 T cells, comprising a mutation in one or more endogenous genes encoding CD58. In some aspects, the cell has reduced or no expression of CD58 protein relative to an unmodified y5 T cell. Some aspects of the present disclosure are directed to modified y5 T cells, comprising a deletion of one or more endogenous genes encoding CD58, wherein the cell has reduced or no expression of CD58 protein relative to an unmodified y5 T cell. In some aspects, the modified y5 T cells express B2M. In some aspects, the modified y5 T cells express CIITA. In some aspects, modified y5 T cells (i) have reduced expression of CD58 protein relative to an unmodified y5 T cell, (ii) express B2M, and (iii) express CIITA. In some aspects, modified y5 T cells (i) have no detectable expression of CD58 protein, (ii) express B2M, and (iii) express CIITA. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous CIITA gene. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous B2M gene. In some aspects, the modified y5 T cells do not comprise a mutation or deletion in an endogenous CIITA gene or an endogenous B2M gene. In some aspects, the modified y5 T cells do not comprise a deletion in an endogenous CIITA gene or an endogenous B2M gene. In some aspects, the modified y5 T cells do not comprise a mutation in an endogenous CIITA gene or an endogenous B2M gene.

[0098] In some aspects, the expression of the CD58 is less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% the CD58 expression in a wild-type cell of the same cell type. In some aspects, the cell has no detectable expression of the CD58.

[0099] Any methods can be used to reduce or eliminate the endogenous expression of B2M, CIITA, or CD58. In some aspects, the reduced expression or deletion of the endogenous gene results from a mutation in, insertion of a transgene into or a deletion (knock-out) of the endogenous gene. In some aspects, the mutation in, or a deletion of, the one or more endogenous genes comprises a mutation in the one or more endogenous genes that results in a loss of expression of a protein encoded by the endogenous gene. In some aspects, the mutation in, or a deletion of, the one or more endogenous genes comprises a deletion in the one or more endogenous genes that results in a loss of expression of a protein encoded by the endogenous gene.II.A.1. Modified Expression of HLA-E

[0100] In some aspects, the modified y5 T cells further comprises increased expression of HLA-E relative to a wild-type y5 T cell, e.g. , the cell prior to modification. In some aspects, the increased expression of the HLA-E results from transfection of a nucleic acid encoding an HLA-E polypeptide. In some aspects, the increased expression of the HLA-E results from a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide. In some aspects, the increased expression of the HLA-E results from (i) transfection of a nucleic acid encoding an HLA-E polypeptide and (ii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide. In some aspects, the expression of the HLA-E is at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190% at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000% that of the expression of HLA-E in a wild-type y5 T cell.

[0101] In some aspects, the modified y5 T cellfurther comprises HLA-E. In some aspect, the expression of the HLA-E is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% at least about 100% that of the expression of HLA-E in a wild-type y5 T cell.

[0102] In some aspects, the HLA-E is a human HLA-E polypeptide. In some aspects, the HLA-E polypeptide is a chimeric polypeptide comprising a human HLA-E polypeptide or a portion thereof. In some aspects, the HLA-E polypeptide is a chimeric polypeptide comprising a HLA-E polypeptide linked to a human B2M polypeptide. In some aspects, the HLA-E polypeptide comprises an amino acid sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of wild-type human HLA-E.

[0103] In some aspects, the HLA-E is encoded by a nucleotide sequence having 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 96%, at least about 97%, at least about 98%, or at least about 99% identity to the nucleotide sequence set forth in SEQ ID NO: 3 (Table 3). In some aspects, the HLA-E is encoded by a nucleotide sequence set forth in SEQ ID NO: 3 (Table 3).

[0104] In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding B2M, wherein the expression of B2M protein is reduced relative to an unmodified y5 T cell, and (ii) a heterologous nucleic acid encoding an HLA-E polypeptide, wherein the expression of HLA-E is increased relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and CD58. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding B2M, wherein the expression of B2M protein is reduced relative to an unmodified y5 T cell, and (ii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and CD58. In some aspects, the modified y5 T cell (i) has reducedexpression of B2M protein relative to an unmodified y5 T cell, (ii) expresses CIITA, (iii) expresses CD58, and (iv) has increased expression of HLA-E protein relative to an unmodified y5 T cell.

[0105] In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CIITA, wherein the expression of CIITA protein is reduced relative to an unmodified y5 T cell, and (ii) a heterologous nucleic acid encoding an HLA-E polypeptide, wherein the expression of HLA-E is increased relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses B2M and CD58. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CIITA, wherein the expression of CIITA protein is reduced relative to an unmodified y5 T cell, and (ii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses B2M and CD58. In some aspects, the modified y5 T cell (i) has reduced expression of CIITA protein relative to an unmodified y5 T cell, (ii) expresses B2M, (iii) expresses CD58, and (iv) has increased expression of HLA-E protein relative to an unmodified y5 T cell.

[0106] In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CD58, wherein the expression of CD58 protein is reduced relative to an unmodified y5 T cell, and (ii) a heterologous nucleic acid encoding an HLA-E polypeptide, wherein the expression of HLA-E is increased relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and B2M. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CD58, wherein the expression of CD58 protein is reduced relative to an unmodified y5 T cell, and (ii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and B2M. In some aspects, the modified y5 T cell (i) has reduced expression of CD58 protein relative to an unmodified y5 T cell, (ii) expresses CIITA, (iii) expresses B2M, and (iv) has increased expression of HLA-E protein relative to an unmodified y5 T cell.

[0107] In some aspects, the cells disclosed herein have has increased persistence in vivo relative to a wild-type cell of the same cell type. In some aspects, the cell disclosed hereinare less immunogenic when administered to a human subject. In some aspects, the human subject has less of an immune response against the cells following administration. In some aspects, the human subject has reduced NK cell killing of the cells following administration. In some aspects, NK cell mediated killing of the cells following administration is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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%, or at least about 95% relative to a wild-type cell of the same cell type administered to a human subject.Table 3: Coding SequencesII.A.2. Chimeric Antigen Receptors and Exogenous T Cell Receptors

[0108] In some aspects, a modified y5 T cell of the disclosure further comprises a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (eTCR). In some aspects, the y5 T cells are modified to express a CAR. In some aspects, the y5 T cells are modified to express an eTCR. In some aspects, the CAR or the eTCR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the tumor antigen comprises CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA- 125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma- associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-llRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE2, claudin (e g., CLDN18.2) , B7H3 or any combination thereof.

[0109] In some aspects, the y5 T cell comprises a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds mesothelin. In some aspects, the y5 T cell comprises an eTCR, wherein the eTCR comprises an antigen-binding domain that specifically binds mesothelin. In some aspects, the cell comprises an eTCR wherein the eTCR is a gamma-delta TCR. In some aspects, the cell comprises an eTCR wherein the eTCR is a y952TCR.

[0110] In some aspects, the cell comprises a CAR, wherein the CAR comprises an antigenbinding domain that specifically binds CD 19. In some aspects, the cell comprises an eTCR, wherein the eTCR comprises an antigen-binding domain that specifically binds CD 19. In some aspects, the cell comprises an eTCR wherein the eTCR is a gamma-delta TCR. In some aspects, the cell comprises an eTCR wherein the eTCR is a y952TCR.[OHl] In some aspects, the cell comprises a CAR, wherein the CAR comprises an antigenbinding domain that specifically binds BCMA. In some aspects, the cell comprises an eTCR, wherein the eTCR comprises an antigen-binding domain that specifically binds BCMA. In some aspects, the cell comprises an eTCR wherein the eTCR is a gamma-delta TCR. In some aspects, the cell comprises an eTCR wherein the eTCR is a y952TCR.

[0112] In some aspects, the CAR further comprises a 4- IBB and CD3 zeta domain. In some aspects, the y5 T cells comprise a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds a tumor antigen, e.g., human mesothelin or human CD33, and further comprise a 4-1BB and CD3 zeta domain. In some aspects, the CAR further comprises a 4- IBB and CD3 zeta domain.

[0113] In some aspects, the CAR further comprises a hinge region between the antigenbinding domain and the transmembrane domain. In some aspects, the hinge is derived from an immunoglobulin (e.g., derived from hinge regions or loop regions). In certain aspects, these hinges comprise, e.g., IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge regions, fragments thereof (alone or capped by additional sequences, e.g., CHI or CH2 regions sequences), or combinations of fragments from IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge regions. In some aspects, the hinges comprise, e.g., IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM constant domain loop regions, fragments thereof (alone or capped by additional sequences, e.g., from adjacent P-strands), or combinations of fragments from IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, or IgM loop regions. In some aspects, the hinge of the present disclosure comprises hinge region derived sequences, loop region derived sequences, or combinations thereof.

[0114] In some aspects, the hinge region is selected from a CD8 hinge, a CD28 hinge, and an immunoglobulin hinge. In some aspects, the hinge comprises a CD28 hinge.

[0115] In some aspects, the CAR further comprises a transmembrane domain. The transmembrane domain can be derived either from a natural or from a recombinant source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some aspects, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR of the present disclosure has bound to a target.

[0116] In some aspects, a transmembrane domain can include at least the transmembrane region(s) of, e.g., CD8, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55),PSGL1, CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO- 3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, NKG2C, or CD 19.

[0117] In some aspects, the TM domain is derived from CD8, CD2, CD4, CD28, CD45, PD1, CD 152, or any combination thereof.

[0118] In some aspects, the CAR further comprises a costimulatory domain. In some aspects, the costimulatory domain comprises a costimulatory domain of 4-1BB / CD137, interleukin-2 receptor (IL-2R), interleukin- 12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function- associated antigen- 1 (LFA-1), LIGHT, NKG2C, 0X40, DAP 10, B7-H3, CD28 deleted for Lek binding (ICA), BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor 8 chain, a ligand that specifically binds with CD83, or any combination thereof. In some aspects, the CAR comprises a 4- 1BB costimulatory domain.

[0119] In some aspects, the CAR further comprises an intracellular signaling domain. In some aspects, the intracellular signaling domain comprises a CD3 C, activating domain, a CD35 activating domain, a CD3s activating domain, a CD3r| activating domain, a CD79A activating domain, a DAP 12 activating domain, a FCER1G activating domain, a DAP10 / CD28 activating domain, a ZAP70 activating domain, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 C, activating domain.

[0120] In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding B2M, wherein the expression of B2M protein is reduced relative to an unmodified y5 T cell, and (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein; and wherein the modified y5 T cell expresses CIITA and CD58. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding B2M, wherein the expression of B2M protein is reduced relative to an unmodified y5 T cell, (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein, and (ii) a heterologous nucleic acid encoding an HLA-E polypeptide, wherein the expression of HLA-E is increased relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and CD58. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding B2M, wherein the expression of B2M protein is reduced relative to an unmodified y5 T cell, (ii) a nucleic acid molecule encoding a CAR or eTCR disclosedherein, and (iii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and CD58.

[0121] In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CIITA, wherein the expression of CIITA protein is reduced relative to an unmodified y5 T cell, and (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein; and wherein the modified y5 T cell expresses B2M and CD58. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CIITA, wherein the expression of CIITA protein is reduced relative to an unmodified y5 T cell, (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein, and (ii) a heterologous nucleic acid encoding an HLA-E polypeptide, wherein the expression of HLA-E is increased relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses B2M and CD58. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CIITA, wherein the expression of CIITA protein is reduced relative to an unmodified y5 T cell, (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein, and (iii) a modification to an endogenous gene encoding HLA- E polypeptide, wherein the modification increases the expression of the endogenous HLA- E polypeptide relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses B2M and CD58.

[0122] In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CD58, wherein the expression of CD58 protein is reduced relative to an unmodified y5 T cell, and (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein; and wherein the modified y5 T cell expresses CIITA and B2M. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CD58, wherein the expression of CD58 protein is reduced relative to an unmodified y5 T cell, (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein, and (ii) a heterologous nucleic acid encoding an HLA-E polypeptide, wherein the expression of HLA-E is increased relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and B2M. In some aspects, the modified y5 T cell comprises (i) mutation in, or a deletion of, the one or more endogenous genes encoding CD58, wherein the expression of CD58 protein isreduced relative to an unmodified y5 T cell, (ii) a nucleic acid molecule encoding a CAR or eTCR disclosed herein, and (iii) a modification to an endogenous gene encoding HLA- E polypeptide, wherein the modification increases the expression of the endogenous HLA- E polypeptide relative to an unmodified y5 T cell; and wherein the modified y5 T cell expresses CIITA and B2M.

[0123] In some aspects, the y5 T cell is transduced with a viral vector encoding a transgene (e.g., a CAR). In some aspects, the viral vector is a retroviral vector. In some aspects, the viral vector is a lentiviral vector. In some such aspects, the cell may stably express the transgene. In some aspects, the cell may transiently express the transgene.II.A.3. Additional Polypeptides

[0124] In some aspects, the modified cell further overexpresses one or more additional endogenous polypeptides. In some aspects, the modified cell further expresses one or more additional heterologous polypeptides. In some aspects, the modified cell comprises a human interleukin 15 (IL15) polypeptide. In some aspects, the modified cell overexpresses an endogenous IL15 polypeptide. In some aspects, the modified cell expresses a heterologous IL15 polypeptide. In some aspects, the IL15 polypeptide is a membranebound IL 15 polypeptide. In some aspects, the IL 15 polypeptide is a membrane bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra). In some aspects, the IL15 polypeptide comprises an IL15 sushi domain / IL15Ra fusion polypeptide (sushil5). In some aspects, the IL15 polypeptide comprises a membrane bound-IL15 / IL15Ra-LSP fusion (mIL15 / Ra- LSP). In some aspects, the IL15 polypeptide comprises a short IL15 polypeptide (sIL15). In some aspects, the IL15 polypeptide comprises a soluble IL15 polypeptide.

[0125] In some aspects, the modified cell comprises a human chemokine (C-C motif) ligand 19 (CCL19) polypeptide. In some aspects, the modified cell overexpresses an endogenous CCL19 polypeptide. In some aspects, the modified cell expresses a heterologous CCL19 polypeptide.

[0126] In some aspects, the modified cell comprises an HSVTK polypeptide.

[0127] In some aspects, a modified y5 T cell disclosed herein (e.g., expressing a CAR) further expresses an armor protein. In some aspects, the armor protein increases the persistence of the modified immune cell, relative to a similarly modified immune cell that does not express the armor protein. In some aspects, the armor protein increases the proliferation of the modified immune cell, relative to a similarly modified immune cell thatdoes not express the armor protein. In some aspects, the armor protein increases the cytotoxicity of the modified immune cell, relative to a similarly modified immune cell that does not express the armor protein.

[0128] In some aspects, the armor protein comprises a membrane-bound polypeptide. In some aspects, the armor proteins comprises a membrane-bound receptor. In some aspects, the membrane-bound receptor comprises IL-15R (e.g., IL-15RP and IL-15Ra). In some aspects, the membrane-bound receptor comprises IL-2R (e.g., IL-2RP). In some aspects, the membrane-bound receptor comprises a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46). In some aspects, the membrane-bound receptor comprises a cytokine receptor (e.g., IL-12 receptor). In some aspects, the membrane-bound receptor comprises a chemokine receptor (e.g., CCR2 receptor). In some aspects, the armor protein comprises a membrane-bound ligand or cytokine. In some aspects, the membrane-bound ligand or cytokine comprises membrane-bound IL-15, membrane-bound IL-7, membrane-bound CD40L, membrane-bound 4-1BB, membrane-bound 4-1BBL, membrane bound CCL19, or any combination thereof.

[0129] In some aspects, the armor protein comprises a soluble polypeptide. In some aspects, the soluble protein comprises a soluble ligand. In some aspects, the soluble protein comprises a cytokine. In some aspects, the armor protein comprises soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, soluble CCL19, or any combination thereof.

[0130] In some aspects, the armor protein comprises a membrane-bound polypeptide and a soluble polypeptide.

[0131] In some aspects, the armor protein comprises an IL-2RP polypeptide. In some aspects, the armor protein further comprises a signal peptide, wherein the signal peptide is cleaved post translation. Any signal peptide can be used. In some aspects, the armor protein comprises an IL-2RP polypeptide, and the armor protein does not comprise an IL-15Ra polypeptide or a construct comprising an IL-15Ra polypeptide tethered to an IL-15 polypeptide.

[0132] In some aspects, the armor protein comprises an IL-15Ra polypeptide. In some aspects, the armor protein comprises a construct comprising an IL-15Ra polypeptide tethered to an IL- 15 polypeptide. In some aspects, the armor protein comprises (i) an IL- 2RP polypeptide and (ii) an IL-15Ra polypeptide tethered to an IL- 15 polypeptide. In someaspects, the armor protein comprises (i) an IL-2RP polypeptide and (ii) a construct comprising an IL-15Ra polypeptide tethered to an IL- 15 polypeptide.

[0133] In some aspects, the IL-15Ra polypeptide is tethered to the IL- 15 polypeptide by a linker. In some aspects, the linker comprises one or more peptide bonds. In some aspects, the linker comprises a peptide linker. In some aspects, the peptide linker is a flexible linker. In some aspects, the linker is a rigid linker. In some aspects, the linker is a cleavable linker. In some aspects, the linker is a Gly-Ser linker. In some aspects, the linker comprises one or more repeats of the sequence GGGS.

[0134] In some aspects, the construct comprising the IL-15Ra polypeptide is tethered to the IL- 15 polypeptide is arranged from N-terminal to C-terminal according to the following order: (i) N-terminus, (ii) the IL-15 polypeptide, (iii) a peptide linker (e.g., a polypeptide comprising GGGS); (iv) the IL-15Ra polypeptide; (v) C-terminus.

[0135] In some aspects, the armor protein is expressed as a single polypeptide. In some aspects, the armor protein is expressed as a single polypeptide comprising (i) an IL-2RP polypeptide; (ii) a linker; and (iii) an IL-15Ra polypeptide tethered to an IL- 15 polypeptide. In some aspects, the armor protein is expressed as a single polypeptide comprising (i) an IL-2RP polypeptide; (ii) a cleavable linker; and (iii) an IL-15Ra polypeptide tethered to an IL- 15 polypeptide. In some aspects, the cleavable linker comprises a P2A sequence.II.A.4. Cells

[0136] Any type of y5 T cell can be used in the compositions and methods disclosed herein. y5 T cells represent a subset of T cells that express on their surface a distinct, defining y5 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (5) chain. Human y5 T cells can be broadly classified as one or two types — peripheral blood-resident y5 T cells and tissue-resident y5 T cells. Most blood-resident y5 T cells express a V52 TCR, whereas this is less common among tissue-resident y5 T cells, which more frequently use V51 and / or other V5 chains. In some aspects, the y5 T cell is a V51+cell. In some aspects, the y5 T cell is a V52+cell. In some aspects, the y5 T cell is a human y5 T cell. In some aspects, the y5 T cell is obtained from a blood sample obtained from a human donor. In some aspects, the y5 T cell is obtained from a skin sample obtained from a human donor. In some aspects, the skin sample comprises a skin biopsy. In some aspects, the y5 T cell is differentiated from an iPSC.

[0137] In some aspects, the y5 T cells described herein lack a vesicular stomatitis virus G glycoprotein (VSV-G) entry receptor (e.g., LDL). In some aspects, the y5 T cell express ASCT-1 and / or ASCT-2. In some aspects, the expression of ASCT-1 and / or ASCT-2 permits transduction with a betaretroviral pseudotyped vector (e.g., BaEV and RD114). In some aspects, the lack of expression of VSV-G prevents transduction with a VSV-G pseudotyped vector.

[0138] Some aspects of the present disclosure are directed to a population of cells comprising one or more modified y5 T cell disclosed herein. In some aspects, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 96%, at least about 97%, at least about 98%, or at least about 99% of the cells of the population of cells are modified y5 T cells disclosed herein. In other words, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 96%, at least about 97%, at least about 98%, or at least about 99% of cells in a population of cells are modified y5 T cells described herein. In some aspects, at least about 25% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 30% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 35% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 40% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 45% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 50% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 55% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 60% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 65% of the population of cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 70% of the population of cells comprisesthe modified y5 T cells disclosed herein. In some aspects, at least about 75% of the population of cells comprises the modified y5 T cells disclosed herein.

[0139] Some aspects of the present disclosure are directed to a population of y5 T cells comprising one or more modified y5 T cell disclosed herein. In some aspects, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 96%, at least about 97%, at least about 98%, or at least about 99% of the population of y5 T cells are modified y5 T cells disclosed herein. In other words, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 96%, at least about 97%, at least about 98%, or at least about 99% of cells in a population of y5 T cells are modified y5 T cells described herein. In some aspects, at least about 25% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 30% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 35% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 40% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 45% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 50% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 55% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 60% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 65% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 70% of the population of y5 T cells comprises the modified y5 T cells disclosed herein. In some aspects, at least about 75% of the population of y5 T cells comprises the modified y5 T cells disclosed herein.

[0140] In some aspects, the modified y5 T cell has increased persistence in vivo relative to an unmodified y5 T cell. In some aspects, the persistence of the modified y5 T cell isincreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%.III. Methods of the DisclosureIII. A. Methods of Engineering

[0141] Some aspects of the present disclosure are directed to a method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding B2M in a y5 T cell, wherein the expression of B2M protein is reduced relative to the expression of B2M by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of CIITA and CD58.

[0142] Some aspects of the present disclosure are directed to a method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding CIITA in a y5 T cell, wherein the expression of CIITA protein is reduced relative to the expression of CIITA by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CD58.

[0143] Some aspects of the present disclosure are directed to a method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding CD58 in a y5 T cell, wherein the expression of CD58 protein is reduced relative to the expression of CD58 by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CIITA.

[0144] In some aspects, the method further comprises (i) introducing into the cell, a nucleic acid encoding an HLA-E polypeptide; (ii) modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii).

[0145] In some aspects, the one or more endogenous genes are genetically modified using a gene editing tool. Any gene editing tool can be used to make the genetic modifications disclosed herein. In some aspects, the gene editing tool comprises a CRISPR / Cas9, CRISPR / Casl2, TALEN, a zinc-finger endonuclease, or any combination thereof. In some aspects, the one or more endogenous genes are genetically modified by introducing into thecell CRISPR / Cas9 and a polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences within the endogenous gene.

[0146] Some aspects of the present disclosure are directed to methods of engineering a human y5 T cell, comprising: (i) transfecting the y5 T cell with a nucleic acid encoding a CAR or an eTCR; and (ii) mutating or deleting an endogenous genes selected from B2M, CIITA, and CD58, wherein the mutation or deletion results in a reduced expression of B2M, CIITA, or CD58 relative to a wild-type y5 T cell.

[0147] Some aspects of the present disclosure are directed to methods of engineering a human y5 T cell, comprising: (i) transfecting the y5 T cell with a nucleic acid encoding a CAR or an eTCR; and (ii) mutating or deleting one or more endogenous genes encoding B2M, wherein the mutation or deletion results in a reduced expression of B2M relative to a wild-type y5 T cell; and wherein the modified y5 T cell retains expression of CIITA and CD58. In some aspects, the method further comprises (iii) overexpressing in the y5 T cell an HLA-E polypeptide. In some aspects, the method comprises transfecting the y5 T cell with a nucleic acid encoding an HLA-E polypeptide. In some aspects, the method comprises modifying an endogenous gene encoding HLA-E polypeptide in the y5 T cell, wherein the modification increases the expression of the endogenous HLA-E polypeptide.

[0148] Some aspects of the present disclosure are directed to methods of engineering a human y5 T cell, comprising: (i) transfecting the y5 T cell with a nucleic acid encoding a CAR or an eTCR; and (ii) mutating or deleting one or more endogenous genes encoding CIITA, wherein the mutation or deletion results in a reduced expression of CIITA relative to a wild-type y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CD58. In some aspects, the method further comprises (iii) overexpressing in the y5 T cell an HLA-E polypeptide. In some aspects, the method comprises transfecting the y5 T cell with a nucleic acid encoding an HLA-E polypeptide. In some aspects, the method comprises modifying an endogenous gene encoding HLA-E polypeptide in the y5 T cell, wherein the modification increases the expression of the endogenous HLA-E polypeptide.

[0149] Some aspects of the present disclosure are directed to methods of engineering a human y5 T cell, comprising: (i) transfecting the y5 T cell with a nucleic acid encoding a CAR or an eTCR; and (ii) mutating or deleting one or more endogenous genes encoding CD58, wherein the mutation or deletion results in a reduced expression of CD58 relative to a wild-type y5 T cell; and wherein the modified y5 T cell retains expression of CIITA and B2M. In some aspects, the method further comprises (iii) overexpressing in the y5 T cellan HLA-E polypeptide. In some aspects, the method comprises transfecting the y5 T cell with a nucleic acid encoding an HLA-E polypeptide. In some aspects, the method comprises modifying an endogenous gene encoding HLA-E polypeptide in the y5 T cell, wherein the modification increases the expression of the endogenous HLA-E polypeptide.

[0150] In some aspects, the CAR or the eTCR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the tumor antigen comprises CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c- Met, mesothelin, GD3, HERV-K, IL-llRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE, claudin (e g., CLDN18.2), B7H3 or any combination thereof.

[0151] In some aspects, the method further comprises transfecting the cells with a nucleic acid encoding an IL15 polypeptide, e.g., an IL15 polypeptide disclosed herein. In some aspects, the IL15 polypeptide is a membrane-bound IL15 polypeptide. In some aspects, the IL15 polypeptide is a membrane bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra). In some aspects, the IL15 polypeptide comprises an IL15 sushi domain / IL15Ra fusion polypeptide (sushi 15). In some aspects, the IL 15 polypeptide comprises a membrane bound-IL15 / IL15Ra-LSP fusion (mIL15 / Ra-LSP). In some aspects, the IL 15 polypeptide comprises a short IL15 polypeptide (sIL15). In some aspects, the IL15 polypeptide comprises a soluble IL15 polypeptide.

[0152] In some aspects, the method further comprises transfecting the cells with a nucleic acid encoding a CCL19 polypeptide. In some aspects, the exogenous gene is inserted to the site of B2M gene. In some aspects, the exogenous gene is inserted to the site of CIITA gene. In some aspects, the exogenous gene is inserted to the site of CD58 gene.III.B. Methods of Treatment

[0153] Some aspects of the present disclosure are directed to methods of treating a disease or condition in a subject in need thereof comprising administering to the subject a composition disclosed herein. In some aspects, the method comprises administering a modified or engineered y5 T cell disclosed herein. In some aspects, the method comprises administering a population of y5 T cells disclosed herein.

[0154] In some aspects, the disease or condition comprises a cancer, z.e., the subject is afflicted with a cancer. In some aspects, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is relapsed. In some aspects, the cancer is refractory or relapsed following one or more prior anti-cancer therapy. In some aspects, the one or more prior anti-cancer therapy comprises a standard of care therapy.

[0155] In some aspects, the compositions disclosed herein are administered in combination with an additional anti-cancer therapy. In some aspects, the additional anti-cancer therapy comprises a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof. In some aspects, the additional anti-cancer therapy comprises a chemotherapy. In some aspects, the additional anti-cancer therapy comprises an immune- checkpoint inhibitor. In some aspects, the additional anti-cancer therapy comprises a PD-1antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR antagonist, or any combination thereof. In some aspects, the anti -cancer therapy comprises an antibody or antigen-binding portion thereof the specifically binds and inhibits PD-1. In some aspects, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof the specifically binds and inhibits PD-L1.

[0156] In some aspects, the method further comprises pretreating the subject prior to administering the population of immune cells. In some aspects, the subject is administered a chemotherapy prior to administering the population of immune cells. In some aspects, the subject is administered an immuno-depleting chemotherapy prior to administering the population of immune cells. In some aspects, the immuno-depleting chemotherapy comprises cyclophosphamide, fludarabine, or both.

[0157] In some aspects, the method comprises administering to the subject (i) the population of y5 T cells disclosed herein and (ii) a cytokine. In some aspects, the cytokine comprises IL-2, an analog thereof, a variant thereof, or a fragment thereof.III.C. Cell Therapy Products

[0158] Some aspects of the present disclosure are directed to a population of cells comprising a plurality of modified y5 T cells disclosed herein. Some aspects of the present disclosure are directed to a cell therapy comprising a population of modified y5 T cells disclosed herein. In some aspects, the population of y5 T cells is cryopreserved. Any method of cry opreservation of cells, e.g., y5 T cells, can be used in the methods and compositions disclosed herein. In some aspects, the y5 T cells are cryopreserved in the presence of DMSO. In some aspects, the cell therapy is cryopreserved to facilitate shipment of the cells.

[0159] The cell therapy and / or population of y5 T cells disclosed herein can be further formulated with one or more excipient. Any excipient that can preserve the cells can be used in the methods and compositions disclosed herein. In some aspects, the cell therapy and / or population of y5 T cells is formulated with one or more excipient that allows for cry opreservation of the y5 T cells, e.g., DMSO.

[0160] In some aspects, the population of y5 T cells comprises a plurality of modified y5 T cells disclosed herein and one or more additional cell. In some aspects, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at leastabout 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%, or at least about 95% of the cells in the population of y5 T cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 10% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 20% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 25% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 30% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 40% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 50% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 60% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 65% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 70% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 75% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 80% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein. In some aspects, at least about 90% of the cells in the population of cells comprise a plurality of modified y5 T cells disclosed herein.

[0161] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis etal. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986)); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2ndEd. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0162] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0163] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1: Immunogenicity of y6 T Cell

[0164] Successful CAR-T therapy requires expansion of CAR-T cells in vivo after administertion to a patient. In general, adminsitered CAR-T cells show in vivo expansion for about 2 to 3 weeks following transfusion, which can be longer when the CAR-T cells are autologous. Mixed lymphocyte reaction (MLR) evaluation revealed that human leukocyte antigen (HLA) Mismatched PBMC donors have limited alloreactive response to mitomycin-treated V51 y5 T cells. Among the 10 PBMC HLA-mismatched donors, only 2 exhibited proliferation in response to V51 y5 T cells (FIG. 1). However, the degree of alloreactivity varied in response to different y5 T cell donors (FIG. 2). V51 y5 T cell triggered lower alloreactivity as measured by PBMC proliferation in all HLA mismatched PBMC donors, compared to iT (unmodified) cells.

[0165] Though these preliminary data suggest that y5 T cells do not invoke a strong immunogenic response, cell activation status (i.e., based on cell diameter) could influence the immunogenictiy of T cells (FIGs. 3A-3B). This can be due to several factors that affect their ability to recognize, respond to, and amplify immune responses. When aP T cells are activated, they up-regulate co-stimulatory molecules, e.g., CD28, CD40L, ICOS, to enhance the immune response. Activated aP T cells also produce a wide range of cytokines, which help amplify the immune cells, heighten TCR sensitivity, and increase proliferation.The level of PBMC proliferation was measured following culture with donor y5 T cells for about 10 days (FIGs. 3 A and 3B) and aP T cells for 9 days (FIG. 3 A) or 5 days (FIG. 3B).Example 2: Gerneration of Hypoimmunogenic y6 T Cell

[0166] In view of the slight, but nonetheless observed, potential immunogenicity of alogenic y5 T cells, hypoimmune editing strategies of V51 y5 T cells were evaluated using the Donor D cells, which exhibited an immune response. Alloreactive donor D PBMCs cocultured with B2M knock-out (KO) and B2M / CIITA double KO V51 y5 T cells showed reduced proliferation as compared to wild type (FIGs. 4A-4H). Further, V51 y5 T cells having either B2M KO or B2M / CIITA double knock-out (dKO) were more susceptible to killing by natural killer (NK) cells. NK donor cells were co-cultured with y5 T cells. For two NK cell donors, the percent of target death CTV+ of B2M KO and DKO y5 T cells was higher than unmodified y5 T cells (FIGs. 5 A-5B). While still reducing PBMC proliferation (FIG. 6A), the increased killing by NK cells was partially reversed in both single and double KO y5 T cells that were further modified to have increased expression of HLA-E (FIGs. 6B-6C). Further expression of a dominant negative FAS by the KO and DKO y5 T cells further improved survival slightly in some samples, whereas y5 T cells having a single knock out of CIITA showed a reduction in T cell alloreactivity without inducing NK killing (FIGs. 6A-6C).

[0167] These experiments were repeated using y5 T cells modified to knock out CD58 or HLA-A. Single knock out of CD58 and HLA-A had less impact on cell exapnasion than observed with B2M KO (FIG. 7A); however, both CD58 KO and HLA-A KO y5 T cells reduced proliferation of the PBMC D cells (FIG. 7C), and CD58 KO y5 T cells did not induce NK killing (FIGs. 7D-7E).***

[0168] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0169] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undueexperimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0170] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0171] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

[0172] While various specific aspects have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

1. WHAT IS CLAIMED:

1. A modified gamma delta (y5) T cell, comprising a mutation in, or a deletion of, one or more endogenous genes encoding beta-2-microglobulin (B2M), wherein the cell has reduced or no expression of B2M protein relative to an unmodified y5 T cell.

2. The modified y5 T cell of claim 1, wherein the y5 T cell expresses class II major histocompatibility complex transactivator (CIITA).

3. The modified y5 T cell of claim 1 or 2, wherein the y5 T cell expresses CD58.

4. A modified y5 T cell, comprising a mutation in, or a deletion of, one or more endogenous genes encoding CIITA, wherein the cell has reduced or no expression of CIITA protein relative to an unmodified y5 T cell.

5. The modified y5 T cell of claim 4, wherein the y5 T cell expresses B2M.

6. The modified y5 T cell of claim 4 or 5, wherein the y5 T cell expresses CD58.

7. A modified y5 T cell, comprising a mutation in, or a deletion of, one or more endogenous genes encoding CD58, wherein the cell has reduced or no expression of CD58 protein relative to an unmodified y5 T cell.

8. The modified y5 T cell of claim 7, wherein the y5 T cell expresses B2M.

9. The modified y5 T cell of claim 7 or 8, wherein the y5 T cell expresses CIITA.

10. The modified y5 T cell of any one of claims 1 to 9, further comprising an increased expression of HLA-E relative to an unmodified y5 T cell.

11. The modified y5 T cell of claim 10, wherein the increased expression of the HLA-E results from:(i) a transfection of a nucleic acid encoding an HLA-E polypeptide;(ii) a modification to an endogenous gene encoding HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or(iii) both (i) and (ii).

12. The modified y5 T cell of any one of claims 1 to 11, wherein the mutation in, or a deletion of, the one or more endogenous genes comprises a mutation in the one or more endogenous genes that results in a loss of expression of a protein encoded by the endogenous gene.

13. The modified y5 T cell of any one of claims 1 to 11, wherein the mutation in, or a deletion of, the one or more endogenous genes comprises a deletion in the one or more endogenous genes that results in a loss of expression of a protein encoded by the endogenous gene.

14. The modified y5 T cell of any one of claims 1 to 13, which is a V51+cell.

15. The modified y5 T cell of any one of claims 1 to 13, which is a V52+cell.

16. The modified y5 T cell of any one of claims 1 to 15, which is a human y5 T cell.

17. The modified y5 T cell of any one of claims 1 to 16, which is obtained from blood or skin.

18. The modified y5 T cell of any one of claims 1 to 17, further comprising a chimeric antigen receptor (CAR) or engineered T cell receptor (eTCR).

19. The modified y5 T cell of claim 18, wherein the CAR or the eTCR comprises an antigenbinding domain that specifically binds a tumor antigen.

20. The modified y5 T cell of claim 19, wherein the tumor antigen comprises CD 19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c- Met, mesothelin, GD3, HERV-K, IL-llRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE, claudin, B7H3 or any combination thereof.

21. The modified y5 T cell of any one of claims 1 to 20, comprising a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds mesothelin.

22. A method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding B2M in a y5 T cell, wherein the expression of B2M protein is reduced relative to the expression of B2M by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of CIITA and CD58.

23. A method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding CIITA in a y5 T cell, wherein the expression of CIITA protein is reduced relative to the expression of CIITA by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CD58.

24. A method of engineering a hypoimmunogenic y5 T cell, comprising genetically modifying one or more endogenous genes encoding CD58 in a y5 T cell, wherein the expression of CD58 protein is reduced relative to the expression of CD58 by an unmodified y5 T cell; and wherein the modified y5 T cell retains expression of B2M and CIITA.

25. The method of any one of claims 22 to 24, further comprising(i) introducing into the cell, a nucleic acid encoding an HLA-E polypeptide;(ii) modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or(iii) both (i) and (ii).

26. The method of any one of claims 22 to 25, wherein the one or more endogenous genes are genetically modified using a gene editing tool.

27. The method of claim 26, wherein the gene editing tool comprises a CRISPR / Cas9, CRISPR / Casl2, TALEN, a zinc-finger endonuclease, or any combination thereof.

28. The method of claim 26 or 27, wherein the one or more endogenous genes are genetically modified by introducing into the cell CRISPR / Cas9 and a polynucleotide sequence encoding one or more guide sequences that hybridize to one or more target sequences within the endogenous gene.

29. The method of any one of claims 22 to 28, wherein the y5 T cell is a V51+cell.

30. The method of any one of claims 22 to 28, wherein the y5 T cell is a V52+cell.

31. The method of any one of claims 22 to 30, wherein the y5 T cell is a human y5 T cell.

32. The method of any one of claims 22 to 31, wherein the y5 T cell is obtained from blood or skin.

33. The method of any one of claims 22 to 32, further comprising introducing into the y5 T cell, a nucleic acid encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR).

34. The method of claim 33, wherein the CAR or the eTCR comprises an antigen-binding domain that specifically binds a tumor antigen.

35. The method of claim 34, wherein the tumor antigen comprises CD 19, CD20, R0R1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-llRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gplOO, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, BCMA, GCC, ADGRE, claudin or any combination thereof.

36. The method of claim 35, wherein the tumor antigen comprises mesothelin.

37. The method of any one of claims 22 to 36, further comprising transfecting the y5 T cell with a nucleic acid encoding an interleukin 15 (IL 15) polypeptide.

38. The method of claim 37, wherein the IL15 polypeptide is a membrane bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra).

39. A modified y5 T cell prepared according to the method of any one of claims 22 to 38.

40. The modified y5 T cell of any one of claims 1 to 21 and 39, which has increased persistence in vivo relative to an unmodified y5 T cell.

41. A population of cells comprising the modified y5 T cell of any one of claims 1 to 21, 39, and 40.

42. A cell population, wherein at least 50% of the cells in the population comprises a modified y5 T cell of any one of claims 1 to 21, 39, and 40.

43. A method of treating a subject in need thereof, comprising administering to the subject the modified y5 T cell of any one of claims 1 to 21, 39, and 40 or the population of cells of claim 41 or 42.

44. The method of claim 43, wherein the subject is afflicted with a cancer.

45. The method of claim 44, wherein the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof.

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