Modified cells with low immunogenicity

Abstract

The present disclosure relates to engineered cells, such as chimeric antigen receptor (CAR) T cells and / or exogenous T cell receptor (eTCR) cells, that have reduced immunogenicity in vivo, and uses thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 371,060, filed August 10, 2022, which is incorporated herein by reference in its entirety.

[0002] Referencing sequence listings submitted electronically via EFS-WEB The contents of the sequence listing submitted electronically in this application (Name: 3817_134PC01_SequenceListing_ST26.xml, Size: 69,591 bytes; and Creation Date: August 9, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to engineered cells, such as chimeric antigen receptor (CAR) immune cells and / or exogenous T cell receptor (eTCR) immune cells, that have reduced immunogenicity in vivo, and uses thereof. [Background technology]

[0004] Cell-based immunotherapy is a rapidly developing field of research aimed at developing new and improved treatments for various diseases, including cancer. Various therapies have been approved, including isolating T cells from a subject, modifying them to express a chimeric antigen receptor (CAR) or an exogenous T cell receptor (eTCR) that can direct the T cells to a specific antigen, and then administering the modified T cells back into the same subject. While autologous CAR-T or eTCR therapy has proven very promising, this process can be time-consuming, expensive, and produce inconsistent results, at least in part due to donor variability.

[0005] One way to streamline the process is to create "off-the-shelf" modified immune cells, which are derived from induced pluripotent stem cells (iPSCs). However, administering HLA-incompatible iPSC-derived immune cells to patients can cause immune responses in the patient, leading to the patient's rejection of the cell therapy. Thus, there remains a need in the art for the development of iPSC-derived cell therapies that can avoid the patient's immune response. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides, at least in part, novel methods and modified cells that can avoid killing by a patient's immune system when the cells (e.g., populations of immune cells or induced pluripotent cells transduced with a CAR or TCR and containing specific modifications described herein) are administered to a patient as a cell therapy formulation. The invention is based, at least in part, on the discovery that expressing Fas dominant-negative (Fas-DN) and HLA-E in cells (e.g., immune cells) while reducing expression of endogenous MHC-I HLA and MHC-II HLA not only avoids natural killer (NK) cell-mediated killing and T cell-mediated killing, but also surprisingly avoids Fas-mediated apoptosis, thereby resulting in cells that can persist longer in vivo. Such a combination of modifications helps to remove a major obstacle to off-the-shelf immune cell therapies.

[0007] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (eTCR); (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) a polypeptide comprising dominant-negative Fas (Fas-DN), Fas-CD27 chimeric polypeptide (Fas-CD27), Fas-4-1BB chimeric polypeptide (Fas-BB), Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

[0008] In some embodiments, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In some embodiments, the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. In some embodiments, the reduced expression of the MHC-I human leukocyte antigens is due to a mutation or deletion of one or more endogenous genes encoding beta-2-microglobulin (B2M). In some embodiments, the reduced expression of the endogenous MHC-II human leukocyte antigens is due to a mutation or deletion of one or more endogenous genes encoding class II major histocompatibility complex transactivator (CIITA).

[0009] In some embodiments, the cells further comprise reduced expression of endogenous CD58 compared to wild-type cells of the same cell type. In some embodiments, the reduced expression of CD58 is due to mutation or deletion of one or more endogenous genes encoding CD58. In some embodiments, the cells comprise a nucleic acid encoding Fas-DN. In some embodiments, the cells comprise a nucleic acid encoding Fas-CD27. In some embodiments, the cells comprise a nucleic acid encoding Fas-BB. In some embodiments, the cells comprise a nucleic acid encoding Fas-OX40.

[0010] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-DN.

[0011] Some embodiments of the present disclosure are directed to cells comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-CD27.

[0012] Some embodiments of the present disclosure are directed to cells comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-BB.

[0013] Some embodiments of the present disclosure are directed to cells comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-OX40.

[0014] In some embodiments, the cells further comprise reduced expression of endogenous CD58 relative to a wild-type cell of the same cell type, hi some embodiments, the reduced expression of CD58 is due to a mutation or deletion of one or more endogenous genes encoding CD58.

[0015] In some embodiments, the cells further comprise reduced expression of endogenous poliovirus receptor (PVR) relative to a wild-type cell of the same cell type, hi some embodiments, the reduced expression of PVR results from a mutation or deletion in one or more endogenous genes encoding PVR.

[0016] In some embodiments, the cells further comprise increased expression of endogenous HLA-E compared to a wild-type cell of the same cell type. In some embodiments, the increased expression of HLA-E results from one of: (i) introducing into the cell a nucleic acid encoding an HLA-E polypeptide; and (ii) modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii). In some embodiments, the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a B2M polypeptide.

[0017] In some embodiments, the method further comprises transfecting the cell with a nucleic acid encoding an interleukin-15 (IL15) polypeptide. In some embodiments, the IL15 polypeptide is a membrane-bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra).

[0018] In some embodiments, the method further comprises introducing into the cell a heterologous nucleic acid encoding a human chemokine (CC motif) ligand 19 (CCL19) polypeptide.

[0019] In some embodiments, the cells are immune cells, induced pluripotent stem cells (iPSCs), or cells differentiated from iPSCs. In some embodiments, the cells are immune cells or hematopoietic stem cells differentiated from iPSCs. In some embodiments, the cells comprise T cells, NK cells, NKT cells, or tumor-infiltrating lymphocytes.

[0020] In some embodiments, the CAR or eTCR comprises an antigen binding domain that specifically binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, 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 gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-1, IL-1, IL-2 ... -11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, BCMA, GCC, ADGRE, claudins, or any combination thereof.

[0021] Some aspects of the present disclosure are directed to cells prepared according to the methods disclosed herein, in some aspects, the cells have increased persistence in vivo compared to wild-type cells of the same cell type.

[0022] Some aspects of the present disclosure are directed to populations of cells comprising the cells disclosed herein.

[0023] Some embodiments of the present disclosure are directed to a cell population, wherein at least 50% of the cells within the population comprise a cell disclosed herein.

[0024] Some aspects of the present disclosure are directed to methods of treating a subject in need thereof, comprising administering to the subject a cell or cell population disclosed herein.

[0025] In some embodiments, the subject is afflicted with cancer, including bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, cancers, including hematologic malignancies, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof.

[0026] Some embodiments of the present disclosure are directed to a guide RNA capable of hybridizing to a human CD58 gene, comprising a nucleic acid sequence selected from SEQ ID NOs: 1-20 and 41. In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the guide RNA consists of the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the guide RNA consists of the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the guide RNA consists of the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the guide RNA consists of the nucleic acid sequence set forth in SEQ ID NO: 4.

[0027] Some embodiments of the present disclosure are directed to methods of inactivating the human CD58 gene in a cell, comprising contacting the cell with a CD58 guide RNA or a nucleic acid encoding the guide RNA as disclosed herein and a DNA endonuclease or a nucleic acid encoding the DNA endonuclease. In some embodiments, the DNA endonuclease comprises CRISPR / Cas9.

[0028] Some embodiments of the present disclosure are directed to a dominant-negative Fas (Fas-DN) comprising the amino acid sequence set forth in SEQ ID NO:27.

[0029] Some embodiments of the present disclosure are directed to a Fas-CD27 chimeric polypeptide (Fas-CD27) comprising the amino acid sequence set forth in SEQ ID NO:22.

[0030] Some embodiments of the present disclosure are directed to a Fas-4-1BB chimeric polypeptide (Fas-4-1BB) comprising the amino acid sequence set forth in SEQ ID NO:23.

[0031] Some embodiments of the present disclosure are directed to a Fas-OX40 chimeric polypeptide (Fas-OX40) comprising the amino acid sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0032] In some embodiments, the eTCR is a gamma-delta TCR. In some embodiments, the gamma-delta TCR is a Vgamma9-Vdelta2 TCR (g9d2 TCR).

[0033] Some embodiments of the present disclosure are directed to modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iii) a nucleic acid encoding Fas (Fas-DN), increased expression of HLA-E compared to wild-type cells of the same cell type, a nucleic acid encoding a suicide gene, or any combination thereof.

[0034] Some embodiments of the present disclosure are directed to modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); and (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0035] Some embodiments of the present disclosure are directed to modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; and (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0036] Some embodiments of the present disclosure are directed to methods of engineering human cells comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, where the inactivation results in reduced expression of the MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigens, where the inactivation results in reduced expression of the MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iii) transfecting the cells with a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0037] Some embodiments of the present disclosure are directed to methods of engineering human cells comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, where the inactivation results in decreased expression of MHC-I human leukocyte antigens compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigens, where the inactivation results in decreased expression of MHC-II human leukocyte antigens compared to wild-type cells of the same cell type; (iii) inactivating one or more endogenous genes encoding CD58, where the inactivation results in decreased expression of CD58 compared to wild-type cells of the same cell type; and (iv) transfecting the cells with a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof. [Brief explanation of the drawings]

[0038] [Figure 1-1] Figures 1A-1D provide a comparison of the cell surface phenotype (CTV-CD25+) of HLA-mismatched peripheral blood mononuclear cells ("PBMCs"; Figures 1A-1B) and HLA-matched PBMCs (Figures 1C-1D) co-cultured with either wild-type ("WT") meso-induced chimeric antigen receptor ("iCAR")-T cells (Figures 1A and 1C) or MHC-I / MHC-II double knockout ("dKO") meso-iCAR-T (Figures 1B and 1D), as analyzed by flow cytometry. [Figure 1-2] Figure 1E is a bar graph showing the percentage of CD25+ dividing T cells from HLA-matched or HLA-mismatched donors after co-culture with WT meso-iCAR-T cells or MHC-I / MHC-II dKO meso-iCAR-T cells. [Figure 2]1 is a bar graph showing the cytotoxic effect of alloreactive ("allo")-T cells against meso-iCAR-T cells that are (i) edited (WT); (ii) B2M / CIITA double KO; (iii) HLA-ABC / CIITA double KO; or (iv) HLA-ABC / CIITA double KO that additionally overexpress HLA-G and HLA-E. Cells were incubated for 24 hours at effector:target (ET) ratios of 2:1 and 1:1, as indicated. [Figure 3A] These are line graphs showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (Donor 1: Figures 3A-3D). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO (3A). Cells were incubated for 24 hours at effector:target (E:T) ratios of 5:1, 2:1, and 0:1. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3B] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 1: Figure 3B). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and PVR knockout (Figure 3B). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3C]This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 1: Figure 3C). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and CD58 knockout (Figure 3C). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3D] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 1:3D). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO, PVR knockout, and CD58 knockout (Figure 3D). Cells were incubated for 24 hours at effector:target (E:T) ratios of 5:1, 2:1, and 0:1. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3E] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 2: Figure 3E). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO (Figure 3E). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3F]This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 2: Figure 3F). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and PVR knockout (Figure 3F). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3G] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 2: Figure 3G). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and CD58 knockout (Figure 3G). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3H] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 2: Figure 3H). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO, PVR knockout, and CD58 knockout (Figure 3H). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3I]This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 3: Figure 3I). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO (Figure 3I). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3J] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 3: Figure 3J). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and PVR knockout (Figure 3J). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3K] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 3: Figure 3K). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and CD58 knockout (Figure 3K). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3L]This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 3: Figure 3L). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO, PVR knockout, and CD58 knockout (Figure 3L). Cells were incubated for 24 hours at effector:target (E:T) ratios of 5:1, 2:1, and 0:1. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3M] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 4: Figure 3M). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO (Figure 3M). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3N] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 4: Figure 3N). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and PVR knockout (Figure 3N). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3O]This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 4: Figure 3O). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO and CD58 knockout (Figure 3O). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 3P] This is a line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from four donors (donor 4: Figure 3P). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cells. Meso-iCAR-T cells were DKO, PVR knockout, and CD58 knockout (Figure 3P). Cells were incubated at effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for 24 hours. The percentage of dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. [Figure 4]This is a line graph showing the efficacy of meso-iCAR-T cells in NK cell-mediated killing. Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before 24-hour co-incubation with NK cell lines at effector:target (E:T) ratios of 4:1, 2:1, or 1:1. The percentage of meso-iCAR-T dead cells was assessed by FACS and analyzed using FLOWJO software. "DKO" refers to B2M / CIITA double knockout. "TKO" refers to B2M / CIITA / CD58 triple knockout. "+E" refers to additional overexpression of HLA-E. "+F" refers to additional expression of FasDN. "+FasBB" refers to additional expression of the Fas-41BB switch receptor. "+HSVTK" refers to additional expression of HSVTK. [Figure 5](A) Line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from three donors (Donor 1: Figure 5A). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cell lines at effector:label (E:T) ratios of 5:1 and 1.5:1 for 24 hours. The percentage of meso-iCAR-T dead cells was assessed by FACS and analyzed using FLOWJO software. (B) Line graph showing the effect of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from three donors (Donor 2: Figure 5B). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cell lines at effector:label (E:T) ratios of 5:1 and 1.5:1 for 24 hours. The percentage of meso-iCAR-T dead cells was assessed by FACS and analyzed using FLOWJO software. (C) Line graph showing the efficacy of NK cell-mediated meso-iCAR-T cell killing. NK cells were obtained from three donors (donor 3: Figure 5C). Meso-iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-incubation with NK cell lines at effector:label (E:T) ratios of 5:1 and 1.5:1 for 24 hours. The percentage of meso-iCAR-T dead cells was assessed by FACS and analyzed using FLOWJO software. [Figure 6] This is a line graph showing the tumor cell killing efficacy of meso-iCAR-T cells. Meso-iCAR-T cells were co-cultured with GSU tumor cells at effector:target (E:T) ratios of 10:1, 3:1, 1:1, or 0.3:1 for 24 hours. The number of viable cells was assessed by the Cell Titer-Glo luminescent viability assay, which results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The luminescent signal was measured using a Pherastar plate reader. [Figure 7]Line graphs depicting NK cell-mediated killing of meso-iCAR-T cells over time. (A) Normalized time course is shown for NSG-MHC I / II DKO animals initially receiving 1.5 x 10 NK cells, followed by administration of WT (wild-type, positive control), B2M and CIITA KO (untransfected or UTD), B2M / CIITA KO with HLA-E and FasDN overexpression, HLA-ABC KO with HLA-G overexpression, or HLA-ABC knockout with HLA-E overexpression and FasDN iCAR T cells. iCAR T cells were administered at a dose of 1 x 10 cells per animal. (B) Normalized time course is shown for NSG-MHC I / II DKO animals initially receiving 2.0 x 10 NK cells from two donors. One hour later, animals were treated with WT (wild-type), B2M / CIITA KO (untransfected or UTD), B2M / CIITA KO with HLA-E and FasDN overexpression, B2M / CIITA / PVR KO with HLA-E and FasDN overexpression, B2M / CIITA / CD58 KO with HLA-E and FasDN overexpression, or B2M / CIITA / CD58 / PVR KO with HLA-E overexpression and FasDN iCART cells at a dose of 1 x 10 cells per animal. (C) Normalized time course of initial administration of 2.0 x 10 NK cells from two donors to NSG-MHC I / II DKO animals is shown. One hour later, animals were administered WT (wild-type), B2M / CIITA KO (untransfected or UTD), B2M / CIITA KO with overexpression of HLA-E and FasDN, B2M / CIITA / PVR KO with overexpression of HLA-E and FasDN, B2M / CIITA / CD58 KO with overexpression of HLA-E and FasDN, or B2M / CIITA / CD58 / PVR KO with overexpression of HLA-E and FasDN and FasDN iCART cells at a dose of 1 x 106 cells per animal. [Figure 8A]Figure 1 is a line graph showing the persistence of iCART cells over time. NK cell-mediated killing of iCART cells that were further engineered to overexpress the FasBB and / or HSVTK killing switch is shown. NSG-MHCI / II DKO animals were initially administered 2.0 x 10 NK cells. One hour later, animals were treated with WT (wild type); B2M / CIITA KO (untransfected or UTD; DKO-UTD); B2M / CIITA / PVR KO (TKO-UTD); B2M / CIITA / PVR KO (TKO) with overexpression of HLA-E and FasDN; B2M / CIITA / CD58 KO (TKO) with overexpression of HLA-E and HSVTK; B2M / CIITA / PVR KO (TKO) with overexpression of HLA-E, FasDN, and HSVTK; or B2M / CIITA / PVR KO (TKO) with overexpression of HLA-E, FasBB, and HSVTK, each at a dose of 1 × 10 cells per animal. [Figure 8B] This is a line graph showing iCAR T cell persistence over time. Data from an alloreactive T cell rejection assay are shown. NSG-MHCI / II DKO mice were intraperitoneally injected with 200 μL of freshly prepared alloreactive T cells. One hour later, mice were injected with 200 μL of iCAR T cells (WT, DKO-UTD, DKO with HLA-E and FASDN overexpression; PVR KO with HLA-E and FASDN overexpression; CD58 KO with HLA-E and FASDN overexpression; or CD58, PVR KO, HLA-E, and FASDN overexpression). Animals were imaged for bioluminescence signal on a VIS Spectrum on days 1, 3, and 6. This signal was then quantified as total flux (photons / second) within a region of interest (ROI). [Figure 8C]Figure 1 shows a line graph depicting iCART cell persistence over time. GSU tumor cell killing efficacy. NSG-MHC I / II DKO mice were first treated with 1 x 10 luciferase-labeled GSU cells, followed by WT, B2M / CIITA KO, B2M / CIITA KO with HLA-E and FasDN overexpression, or B2M / CIITA / CD58 KO with HLA-E and FasDN overexpression at a dose of 1 x 10 per animal. [Figure 8D] Figure 1 shows a line graph depicting iCART cell persistence over time. GSU tumor cell killing efficacy. NSG-MHC I / II DKO mice were first treated with 1 x 10 luciferase-labeled GSU cells, followed by WT, B2M / CIITA KO, B2M / CIITA KO with HLA-E and FasDN overexpression, or B2M / CIITA / CD58 KO with HLA-E and FasDN overexpression at a dose of 1 x 10 per animal. [Figure 9] (A) Schematic of the use of the Fas receptor in engineered immune cells to affect apoptosis. (B) Schematic of the use of the Fas receptor in engineered immune cells to affect apoptosis. [Figure 10-1] 10A-10D are graphs showing cell proliferation of cells expressing mIL15 / Ra(5BB) (FIG. 10A), dnFAS (FIG. 10B), FAS-4-1BB (FIG. 10C), and FAS-OX40 (FIG. 10D) after stimulation with FAS superligand. [Figure 10-2] Figure 10E is a bar graph showing the fold change in cell number after addition of SuperFas-Ligand at 0.1 ng / ml, 1 ng / ml, 10 ng / ml, and 100 ng / ml. Figure 10F shows the fold expansion during activation in iCAR T cells and cells transduced with 5BB, dnFAS, FAS-4-1BB, and FAS-OX40 constructs. [Figure 11](A) Line graph showing cell proliferation for (i) non-transformed cells prepared with boost and no-boost protocols (UTD, UTD-Boost), (ii) iCAR T cells expressing 5BB prepared with boost and no-boost protocols (5BB, 5BB-Boost), (iii) dnFAS / 5BB, and (iv) FAS-OX40 / 5BB. (B) Line graph showing percentage of target cell lysis (Figure 11B) for (i) non-transformed cells prepared with boost and no-boost protocols (UTD, UTD-Boost), (ii) iCAR T cells expressing 5BB prepared with boost and no-boost protocols (5BB, 5BB-Boost), (iii) dnFAS / 5BB, and (iv) FAS-OX40 / 5BB. [Figure 12A] This is a line graph showing in vivo efficacy data for modified iCART cells in a mouse model. GSU-RFluc cells were intraperitoneally transferred into NSG-MHCI / II DKO mice at a dose of 1 x 10 cells per mouse. On day 4, iCAR-T cells were transferred at a dose of 1 x 10 cells per mouse, and cytolysis was analyzed based on mean fluorescence intensity. Cytolysis over time is shown in seven test groups (PBS, UTD + IL-15, BBp + IL-15, BB-FasDN + IL-15, BB-Fas / 41BB + IL-15, BB-Fas / CD27 + IL-15, and BB-Fas / Ox40 + IL-15). [Figure 12B]

[0033] Figure 1 is a line graph showing in vivo efficacy data for modified iCART cells in a mouse model. GSU-RFluc cells were intraperitoneally transferred into NSG-MHCI / II DKO mice at a dose of 1 x 10 cells per mouse. On day 4, iCAR-T cells were transferred at a dose of 1 x 10 cells per mouse, and cytolysis was analyzed based on mean fluorescence intensity. Comparison of individual animal responses to PBS vs. 5BB is shown. [Figure 12C]This is a line graph showing in vivo efficacy data for modified iCART cells in a mouse model. GSU-RFluc cells were intraperitoneally transferred into NSG-MHCI / II DKO mice at a dose of 1 x 10 cells per mouse. On day 4, iCAR-T cells were transferred at a dose of 1 x 10 cells per mouse, and cytolysis was analyzed based on mean fluorescence intensity. A comparison of individual animal responses to PBS versus FAS-Ox40 is shown. [Figure 13A] 13A and 13B are graphs showing flow cytometry of HLA-E expression in HLA-E knock-in iPS cells (FIG. 13A) and wild-type cells (FIG. 13B). [Figure 13B] 13A and 13B are graphs showing flow cytometry of HLA-E expression in HLA-E knock-in iPS cells (FIG. 13A) and wild-type cells (FIG. 13B). [Figure 13C] 13C is a graph showing flow cytometry of FASDN expression in FASDN knock-in iPS cells (FIG. 13C) and wild-type cells (FIG. 13D). [Figure 13D] 13C is a graph showing flow cytometry of FASDN expression in FASDN knock-in iPS cells (FIG. 13C) and wild-type cells (FIG. 13D). [Figure 13E] 13E and 13F are graphs showing flow cytometry of tEGFR expression in tEGFR knock-in iPS cells (FIG. 13E) and wild-type cells (FIG. 13F). [Figure 13F] 13E and 13F are graphs showing flow cytometry of tEGFR expression in tEGFR knock-in iPS cells (FIG. 13E) and wild-type cells (FIG. 13F). DETAILED DESCRIPTION OF THE INVENTION

[0039] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (eTCR); (ii) reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) a polypeptide comprising dominant-negative Fas (Fas-DN), Fas-CD27 chimeric polypeptide (Fas-CD27), Fas-4-1BB chimeric polypeptide (Fas-BB), Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

[0040] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-DN.

[0041] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-CD27.

[0042] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-BB.

[0043] Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-OX40.

[0044] In some embodiments, the MHC-I human leukocyte antigen is HLA-A, HLA-B, and HLA-C. In some embodiments, the reduced expression of the MHC-I human leukocyte antigen or the inactivated endogenous gene encoding the MHC-I human leukocyte antigen is due to a mutation, transgene insertion (knock-in), or deletion (knock-out) of one or more endogenous genes encoding beta-2-microglobulin (B2M). In some embodiments, the reduced expression of the MHC-I human leukocyte antigen or the inactivated endogenous gene encoding the MHC-I human leukocyte antigen is due to the insertion (knock-in) of all or part of a transgene encoding HLA-E or a protein comprising HLA-E into one or more endogenous genes encoding beta-2-microglobulin (B2M). In some embodiments, a transgene encoding HLA-E or a protein comprising all or part of HLA-E can be inserted into exon 1, 2, or 3, preferably exon 1, of an endogenous gene encoding beta-2-microglobulin (B2M). In some embodiments, the MHC-II human leukocyte antigen is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, reduced expression of endogenous MHC-II human leukocyte antigen or an inactivated endogenous gene encoding MHC-II human leukocyte antigen results from a mutation, transgene insertion, or deletion (knockout) of one or more endogenous genes encoding class II major histocompatibility complex transactivators (CIITAs). In some embodiments, reduced expression of an MHC-II human leukocyte antigen or an inactivated endogenous gene encoding an MHC-II human leukocyte antigen results from the insertion (knock-in) of all or part of a transgene encoding Fas (e.g., Fas-DN), a suicide gene (e.g., HSV-TK or a mutant thereof), and / or a tag protein (e.g., EGFR, LNGFR, or a mutant thereof) into one or more endogenous genes encoding CIITA.In some embodiments, all or part of a transgene encoding Fas (e.g., Fas-DN), a suicide gene (e.g., HSV-TK or a mutant thereof), and / or a tag protein (e.g., EGFR, LNGFR, or a mutant thereof) can be inserted into exon 1, 2, or 3, preferably exon 3, of an endogenous gene encoding CIITA. The transgene may include genes encoding one or more proteins constituting all or part of Fas (e.g., Fas-DN), a suicide gene (e.g., HSV-TK or a mutant thereof), and / or a tag protein (e.g., EGFR, LNGFR, or a mutant thereof). In some embodiments, reduced expression of CD58 or an inactivated endogenous gene encoding CD58 results from a mutation, transgene insertion (knock-in), or deletion (knock-out) of one or more endogenous genes encoding CD58. The deletion (knock-out) site of one or more endogenous genes encoding CD58 can be exon 1, 2, and / or 3, preferably exon 3, of the endogenous gene encoding CD58.

[0045] Before describing the present disclosure in further detail, it should be understood that the present disclosure is not limited to the particular compositions or process steps described. As will be apparent to those skilled in the art upon reading this disclosure, the individual embodiments described and illustrated herein comprise discrete elements and features that may be readily separated from or combined with any of the features of several other embodiments without departing from the scope and spirit of the present disclosure. Any depicted method can be carried out in the order of events depicted or in any other logically possible order.

[0046] The headings provided herein are not limitations of the various aspects of the disclosure, which may 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.

[0047] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below, unless expressly defined otherwise herein. Additional definitions are found throughout this application.

[0048] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and fractions thereof, where appropriate (e.g., tenths and hundredths of an integer), unless otherwise indicated.

[0049] Throughout this disclosure, the terms "a" or "an" refer to one or more entities; for example, "a chimeric polypeptide" is understood to refer to one or more chimeric polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0050] Furthermore, as used herein, "and / or" shall be construed as specifically disclosing the two specified features or components, with or without the other. Thus, the term "and / or," when used in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in phrases such as "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). Furthermore, "or" is used to denote an open list of elements in the list. For example, "X includes A or B" means that X includes A, X includes B, X includes A and B, or X includes A or B and any other element.

[0051] The terms "about" or "essentially consisting of" refer to a value or composition that falls within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which depends in part on the method of measuring or determining the value or composition, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within one standard deviation or more than one standard deviation, as practiced in the art. Alternatively, "about" or "essentially consisting of" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is recited in this application and claims, unless otherwise specified, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable error range for that particular value or composition.

[0052] The terms "activated immune cells," "activated T cells," and "activated NK cells" refer, inter alia, to immune cells, e.g., T cells and / or NK cells, that are undergoing cell division.

[0053] "Antigen" refers to any molecule (e.g., a peptide) that can elicit an immune response or bind to a TCR. The immune response involves antibody production, activation of specific immunocompetent cells, or a combination thereof. One of skill in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Antigens can be endogenously expressed (i.e., expressed by genomic DNA) or recombinantly expressed. Antigens and / or epitopes can be specific to a particular tissue, such as cancer cells, or can be widely expressed. Additionally, fragments of larger molecules can act as antigens. In some embodiments, the antigen is a tumor antigen.

[0054] As used herein, "antigen-presenting cell" or "APC" refers to a cell or cell-like antigen-presenting surface that expresses one or more antigens. In some embodiments, the antigen is presented on the surface of the APC.

[0055] As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in the patient's overall survival or progression-free survival, an increase in the patient's life expectancy, or an improvement in various physiological symptoms of the patient associated with the tumor. An anti-tumor effect can also refer to the prevention of tumor development, for example, a vaccine.

[0056] As used herein, the term "approximately," when applied to one or more values of interest, refers to a value equivalent to a stated reference value. In certain embodiments, unless otherwise specified or clear from the context, the term "approximately" refers to a range of values that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or more in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of possible values).

[0057] The term "autologous" refers to any material (e.g., immune cells) derived from the same individual to which they are subsequently reintroduced. For example, autologous T cell therapy involves administering to a subject T cells isolated from the same subject. The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species. For example, allogeneic T cell transplantation involves administering to a subject T cells obtained from a donor other than the subject.

[0058] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors, which can invade adjacent tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can also include tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, and other white blood cell malignancies. In some embodiments, the methods of the present invention can be used to reduce tumor size, for example, of tumors derived from cancer, including bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, parathyroid cancer, Cancers include renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), or any combination thereof. Certain cancers may be responsive to chemotherapy or radiation therapy or refractory. Refractory cancers refer to cancers that are not amenable to surgical intervention, and such cancers either do not respond to chemotherapy or radiation therapy initially or become unresponsive over time.

[0059] Whenever an embodiment is described herein using the term "comprising," it is understood that otherwise similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0060] As used herein, "cytokine" refers to a non-antibody protein released by a cell in response to contact with a specific antigen, where the cytokine interacts with a second cell and mediates a response in the second cell. Cytokines may be endogenously expressed by the cell, added to cells in culture, administered to a subject, or any combination thereof. Cytokines are released from immune cells (including macrophages, B cells, T cells, and mast cells) to propagate immune responses. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, inflammatory cytokines, effector, and acute phase proteins. For example, homeostatic cytokines (including interleukin (IL) 7 and IL-15) can promote immune cell survival and proliferation, while inflammatory cytokines can promote inflammatory responses. 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 proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, 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 growth factor (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).

[0061] "Chemokines" are a type of cytokine that mediates chemotaxis, or directional movement, of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1a (MIP-1a, MIP-1a), MIP-Ib (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokines (TARC and CCL17).

[0062] 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), CCL13, 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), and oncostatin M. (OSM), CD154, lymphotoxin (LT) beta, 4-IBB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-inducible 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).

[0063] The term "engineered autologous cell therapy," sometimes abbreviated as "eACT™" and also known as adoptive cell transfer, is a process in which a patient's own immune cells (e.g., T cells and / or NK cells) are harvested and then genetically modified to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. The immune cells (e.g., T cells and / or NK cells) can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) immune cells, e.g., T cells or immune cells, are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety containing a costimulatory domain and an activation domain. The costimulatory domain can be derived, for example, from CD28, and the activation domain can be derived, for example, from CD3-zeta (Figure 1). In certain embodiments, CARs are designed to have two, three, four, or more costimulatory domains. CARscFvs can be designed to target, for example, CD19, a transmembrane protein expressed by cells in the B-cell lineage, including all normal B cells and B-cell malignancies (including, but not limited to, NHL, CLL, and non-T-cell ALL). Exemplary CAR-T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference in their entireties.

[0064] As used herein, the term "Fas" refers to the Fas receptor protein or portions thereof. The Fas receptor (also known as Fas, FasR, apoptosis antigen 1, APO-1, APT, CD95, and TNFRSF6) is a cell surface receptor protein for Fas ligand. The canonical amino acid sequence of the human Fas receptor is shown in Table 1 (UniProt P25445, SEQ ID NO: 30). Binding of Fas ligand to the Fas receptor on a cell triggers apoptosis of the cell. Some embodiments of the present disclosure are directed to immune cells engineered to express one or more chimeric polypeptides comprising the extracellular ligand-binding domain of the Fas receptor (Fas ECD) (e.g., SEQ ID NO: 31) linked to a heterologous intracellular domain, e.g., the CD27 intracellular domain (e.g., SEQ ID NO: 22), the 4-1BB intracellular domain (e.g., SEQ ID NO: 23), or the OX40 intracellular domain (e.g., SEQ ID NOs: 24-26). Non-limiting examples of chimeric Fas proteins that can be used in the compositions and methods disclosed herein can be found in Table 1. In some embodiments, the Fas is Fas dominant negative, or "FasDN." As used herein, "FasDN" refers to a Fas receptor that has been modified to contain a truncated Fas intracellular domain. An example of a FasDN sequence that can be used in the compositions and methods disclosed herein is provided in Table 1 (SEQ ID NO:27). In some embodiments, the chimeric Fas polypeptide further comprises a signal peptide. Any signal peptide capable of facilitating expression of the chimeric Fas polypeptide can be used in the compositions and methods disclosed herein. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:29.

[0065] [Table 1-1]

[0066] [Table 1-2]

[0067] "Immune response," as understood in the art, generally refers to a biological reaction in a vertebrate to foreign or abnormal agents, e.g., cancerous cells, which protects the organism from such agents and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body, invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells (e.g., effector T cells, Th cells, CD4 + cells, CD8 + In some embodiments, the immune response refers to NK cell-mediated killing of foreign cells, e.g., allogeneic T cell therapy.

[0068] "Immunotherapy" refers to treating a subject suffering from or at risk of suffering from or recurring with a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune system or immune response.

[0069] As used herein, the term "inactivating" or "inactivation," for example, with respect to a gene or protein, refers to a means capable of inducing reduced expression of the protein. In some embodiments, inactivation can be achieved by deletion or mutation of all or part of the coding region of the gene or all or part of the non-coding region of the gene, resulting in reduced expression of the gene or the protein encoded by the gene. In some embodiments, inactivation is achieved by deletion of the entire coding region of the gene. In some embodiments, inactivation is achieved by partial deletion of the coding region of the gene. In some embodiments, inactivation is achieved by deletion of one or more regulatory elements that drive gene expression. In some embodiments, inactivation is achieved by mutation of one or more regulatory elements, resulting in reduced or lost expression of the gene. In some embodiments, inactivation is achieved by mutation of one or more nucleic acids, resulting in expression of a non-functional protein. In some embodiments, inactivation is achieved by missense mutation, resulting in expression of a non-functional protein. In some embodiments, inactivation is achieved by interference with transcription or translation of the gene, resulting in reduced protein expression. In some embodiments, the reduction in expression is relative to the expression of the target gene in the cell before modification (e.g., deletion or mutation). In some embodiments, expression of the gene is measured before modification, then the cell is modified, and then expression of the gene is measured after modification.

[0070] As used herein, the term "introducing" or "introduction" refers to expressing a heterologous polynucleotide and / or polypeptide in a cell. In some embodiments, introduction is achieved by transfecting the cell with a polynucleotide of interest. In some embodiments, introduction is achieved by genetically engineering the cell to express the heterologous sequence using, for example, but not limited to, gene editing tools including, but not limited to, CRISPR / Cas, CRISPR / Cas9, CRISPR / Cas12, CRISPR / Cas12a, CRISPR / Cpf1, zinc finger, TALEN, Closver-Cas, or variants thereof. In some embodiments, introduction is achieved by contacting the cell with mRNA encoding the polypeptide of interest such that the mRNA enters the cell or the nucleus of the cell. In some embodiments, introduction comprises transfecting or transducing the cell with a polynucleotide encoding the polypeptide.

[0071] As used herein, the term "iPS cell" or "iPSC" refers to a cell that has been dedifferentiated (or reprogrammed) to a more naive state, e.g., a pluripotent state. Various methods for dedifferentiating cells are known, including, but not limited to, overexpressing Oct3 / 4, Sox2, Klf4, and c-Myc ("Yamanaka factors") in the cell (see, e.g., Takahashi and Yamanaka, Cell, 126:663-76 (2006)). In some embodiments, iPS cells are pluripotent, e.g., capable of differentiation into a limited number of cell types. In some embodiments, iPS cells are totipotent, e.g., capable of differentiation into any cell type. In some embodiments, iPS cells can be redifferentiated into specific types of cells, e.g., immune cells.

[0072] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte and a major component of the innate immune system. NK cells eliminate tumor and virus-infected cells by inducing apoptosis, or programmed cell death, in target cells. NK cells have come to be called "natural killers" because they do not require activation to kill target cells. T cells play a major role in cell-mediated immunity. T cell receptors (TCRs) expressed on the surface of T cells distinguish T cells from other lymphocyte types. The thymus is a specialized organ of the immune system and is primarily responsible for T cell maturation. There are six types of T cells: helper T cells (e.g., CD4+ cells); cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells); and memory T cells ((i) stem memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, like naive cells, but express large amounts of CD95, IL-2R.p, CXCR3, and LFA-1, and are memory T cells). (ii) central memory T cells express L-selectin and CCR7 and secrete IL-2, but not IFNγ or IL-4; (iii) effector memory T cells, however, do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ 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.

[0073] B cells (with the participation of antibodies) play a major role in humoral immunity. They produce antibodies and antigens, act as antigen-presenting cells (APCs), and, after activation by antigen interaction, become memory B cells. In mammals, immature B cells are formed in the bone marrow, from which they are named.

[0074] As used herein, "MHC class I molecule" refers to the protein product of a wild-type or mutant HLA class I gene that encodes an MHC class I molecule. Therefore, "HLA class I molecule" and "MHC class I molecule" can be used interchangeably. An MHC class I molecule comprises two protein chains: an alpha chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. The amino acid sequence of β2m is shown in SEQ ID NO: 21 (Table 2). The alpha chain of an 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 over 220 genes of diverse functions. HLA genes are highly mutated, and over 20,000 HLA alleles and associated alleles, including over 15,000 HLA class I alleles, are known in the art, encoding thousands of HLA proteins, including over 10,000 HLA class I proteins. The HLA complex contains at least three genes encoding MHC class I α-chain proteins: HLA-A, HLA-B, and HLA-C. Additionally, HLA-E, HLA-F, and HLA-G encode proteins that associate with MHC class I molecules.

[0075] [Table 2]

[0076] As used herein, "MHC class II molecule" refers to the protein product of a wild-type or mutant HLA class II gene encoding an MHC class II molecule. Therefore, "MHC class II molecule" can be used interchangeably with "HLA class II molecule." A typical MHC class II molecule contains two protein chains: an alpha chain and a beta chain. Generally, naturally occurring alpha and beta chains each contain a transmembrane domain that anchors the alpha / beta chain to the cell surface and an extracellular domain that carries antigen and interacts with TCR and / or CD4 expressed on T cells. 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 over 220 genes with diverse functions. The HLA gene complex is highly variable, with over 20,000 HLA and related alleles, including over 250 MHC class II α-chain alleles and 5,000 MHC class II β-chain alleles known in the art, that encode 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 MHC class II molecules and support their assembly and function.

[0077] As used herein, "pharmaceutically acceptable carriers" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient replenishers, such materials, and combinations thereof, as known to those skilled in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0078] "Enhancing an endogenous immune response" means increasing the efficacy or potency of an existing immune response in a subject. This increased efficacy 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.

[0079] As used herein, the terms "recombinant" or "modified" cells are intended to refer to cells that contain nucleic acids that do not naturally occur within the cell, e.g., immune cells, and can be cells 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. Although certain modifications may occur in progeny, either due to mutation or environmental influences, such progeny are still within the scope of the terms "recombinant" or "modified" as used herein.

[0080] As used herein, the terms "decreased expression" and "increased expression" refer to the expression of a particular gene or protein in a cell compared to a control, e.g., the expression of a particular gene in a modified cell compared to the expression of the gene in a wild-type (unmodified) cell. Relative expression can be based on mRNA and / or protein levels. Any means of measuring mRNA and / or protein levels can be used to determine whether gene or protein expression is decreased or increased, including, but not limited to, immunohistochemistry and PCR-based techniques.

[0081] In some embodiments, cells that have "reduced expression" of a particular gene or protein have an expression level that 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% of the expression of the gene or protein in an unmodified cell, e.g., a wild-type cell of the same cell type. In some embodiments, an unmodified cell, e.g., a wild-type cell of the same cell type, expresses a particular gene or protein, and the modified cell does not have detectable levels of expression of that protein or gene, e.g., is a "knockout" of that gene or protein. Thus, the term "knockout" refers to the complete elimination 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.

[0082] In some embodiments, cells that have "increased expression" or "overexpression" of a particular gene or protein have greater than about 105%, greater than about 110%, greater than about 115%, greater than about 120%, greater than about 125%, greater than about 130%, greater than about 140%, greater than about 150%, greater than about 160%, greater than about 170%, greater than about 180%, greater than about 190%, greater than about 200%, greater than about 225%, greater than about 250%, greater than about 260%, greater than about 270%, greater than about 280%, greater than about 290%, greater than about 300%, greater than about 310%, greater than about 320%, greater than about 330%, greater than about 340%, greater than about 350%, greater than about 360%, greater than about 370%, greater than about 380%, greater than about 390%, greater than about 400%, greater than about 410%, greater than about 425%, greater than about 430%, greater than about 440%, greater than about 450%, greater than about 460%, greater than about 470%, greater than about 480%, greater than about 490%, greater than about 500%, greater than about 510%, greater than about 520%, greater than about 530%, greater than about 540%, greater than about 550%, greater than about 560%, greater than about 570%, greater than about 580%, greater than about 590%, greater than about 600%, greater than about 610%, greater than about 620%, greater than about 630%, greater than about 640%, greater than about 650%, greater than about 660%, greater than about 670%, greater than about 680%, greater than about 690%, greater than about 700%, greater than about The expression level is greater than 75%, greater than about 300%, greater than about 350%, greater than about 400%, greater than about 450%, greater than about 500%, greater than about 600%, greater than about 700%, greater than about 800%, greater than about 900%, or greater than about 1000%, for example, greater than about 200%, greater than about 225%, greater than about 250%, greater than about 275%, greater than about 300%, greater than about 350%, greater than about 400%, greater than about 450%, greater than about 500%, greater than about 600%, greater than about 700%, greater than about 800%, greater than about 900%, or greater than about 1000%, where 100% expression corresponds to the expression observed in wild-type cells.In some embodiments, cells that have "increased expression" or "overexpression" of a particular gene or protein have expression levels that are 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 105% higher, at least about 110% higher, at least about 120% higher, at least about 130% higher, at least about 140% higher, at least about 155% higher, at least about 160% higher, at least about 170% higher, at least about 180% higher, at least about 190% higher, at least about 210% higher, at least about 220% higher, at least about 230% higher, at least about 240% higher, at least about 250% higher, at least about 260% higher, at least about 270% higher, at least about 280% higher, at least about 290% higher, at least about 300% higher, at least about 310% higher, at least about 320% higher, at least about 330% higher, at least about 340% higher, at least about 350% higher, at least about 350% higher, at least about 360% higher, at least about 370% higher, at least about 380% higher, at least about 390% higher, The expression level may be 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 about 350% 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. In some embodiments, an unmodified cell, e.g., a wild-type cell of the same cell type, or the same cell or population of cells prior to modification, does not have expression of a particular gene or protein, and the increased expression is any expression of that gene or protein.

[0083] Increased expression of a particular gene or protein can be achieved by any method. In some embodiments, gene or polypeptide expression is enhanced by introducing a molecule, signal, element, or modification into a cell that results in increased gene or polypeptide expression in the cell. In some embodiments, gene or polypeptide expression is enhanced by transfecting a cell with a nucleic acid molecule encoding the protein. In some embodiments, the nucleic acid of interest is introduced into a cell via electroporation. In some embodiments, the nucleic acid is a vector. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, gene or protein expression is enhanced by modifying endogenous regulatory elements or inserting heterologous regulatory elements into an endogenous gene, thereby increasing the expression of the endogenous gene encoding the polypeptide of interest. In some embodiments, gene or protein expression is enhanced by knocking in a heterologous coding region encoding the polypeptide of interest. In some embodiments, modification of the endogenous sequence is achieved using a gene editing tool such as CRISPR.

[0084] As used herein, the terms "subject" and "patient" are used interchangeably and refer to either a human or a non-human, e.g., a primate, a mammal, and a vertebrate. In certain aspects, the subject is a human.

[0085] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant fusion protein that combines an antigen-specific extracellular domain with an intracellular domain that confers a specific function on the cell upon antigen binding to the extracellular domain. In some aspects, the chimeric antigen receptors disclosed herein comprise a chimeric polypeptide of the present disclosure.

[0086] As used herein, the term "T cell receptor" (TCR) refers to a heteromeric cell surface receptor that can specifically interact 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 their antigen-binding portions, chimeric TCRs, TCR fusion constructs, and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells and are involved in T cell recognition and targeting of antigen-presenting cells. Antigen-presenting cells (APCs) present fragments of foreign proteins (antigens) complexed with major histocompatibility complexes (MHCs; also referred to herein as complexes with HLA molecules, e.g., HLA class 1 molecules). TCRs recognize and bind to the antigen-HLA complex, recruiting CD3 (expressed by T cells) to activate the TCR. Activated TCRs initiate an immune response, including downstream signaling and destruction of antigen-presenting cells.

[0087] As used herein, "foreign TCR" or "eTCR" refers to a TCR that is heterologous to the cell that expresses the TCR. As used herein, the term "heterologous" refers, for example, to something that is not native to a particular cell or that is not found in nature.

[0088] Generally, TCRs can comprise two chains: (i) an alpha chain and a beta chain (alpha-beta TCR) in the case of alpha-beta T cells, and (ii) a gamma chain and a delta chain (gamma-delta TCR) in the case of gamma-delta T cells, which are 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 antigen. The constant region is located proximal to the cell membrane. TCRs can further comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the conventional "constant region," as well as the transmembrane region and cytoplasmic tail, if present.

[0089] The variable domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each alpha and beta chain variable domain contains 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. Although all three CDRs of each chain are involved in antigen binding, CDR3 is considered to be the primary antigen-binding region. CDR1 also interacts with antigen, while CD2 is thought to primarily recognize the ELLA complex. In one embodiment, gamma chain variable domains include Vgamma 1, Vgamma 2, Vgamma 3, Vgamma 4, Vgamma 5, Vgamma 6, Vgamma 7, Vgamma 8, and Vgamma 9, and exemplary delta chain variable domains include Vdelta 1, Vdelta 2, Vdelta 3, Vdelta 4, Vdelta 5, Vdelta 6, Vdelta 7, Vdelta 8, and Vdelta 9. The specific combination of gamma chain variable domain and delta chain variable domain in a TCR is not limited, but for example, the eTCR may be any of Vgamma 3-Vdelta 1 TCR (g3d1 TCR), Vgamma 4-Vdelta 1 TCR (g4d1 TCR), Vgamma 9-Vdelta 1 TCR (g9d1 TCR), and Vgamma 9-Vdelta 2 TCR (g9d2 TCR).

[0090] Unless expressly stated, and unless the context dictates otherwise, the term "TCR" also includes an antigen-binding fragment or portion of any TCR disclosed herein, including monovalent and bivalent fragments or portions, as well as single-chain TCRs. The term "TCR" is not limited to naturally occurring TCRs that bind to the surface of T cells. As used herein, the term "TCR" also 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 has been engineered to express CD3, as described herein), or a TCR described herein that is free from the cell membrane (e.g., an isolated TCR or a soluble TCR).

[0091] "TCR fragment," "antigen-binding molecule," or "portion of a TCR" refers to any portion less than the entire TCR. An antigen-binding molecule can include antigen complementarity-determining regions (CDRs).

[0092] "Suicide gene" refers to a gene that causes a cell to commit suicide. In some embodiments, the suicide gene causes a cell to commit suicide through apoptosis. Non-limiting examples of suicide genes include viral thymidine kinase, cytosine deaminase, intracellular antibodies against antioxidant enzymes (AOEs), bacterial nitroreductase, caspases, and DNases.

[0093] In one embodiment, the suicide gene is a viral thymidine kinase (TK). Thymidine kinase is an ATP-thymidine 5'-phosphotransferase that converts deoxythymidine to deoxythymidine 5'-monophosphate, which is then phosphorylated by viral thymidine kinase and nucleoside diphosphate kinase to deoxythymidine diphosphate and then deoxythymidine triphosphate, respectively. Deoxythymidine triphosphate is incorporated into synthesized DNA molecules by DNA polymerase. Some dNTP analogs, such as ganciclovir (GCV), a synthetic analog of 2'-deoxyguanosine, have the ability to terminate DNA synthesis upon their incorporation into synthesized DNA. Termination of synthesis triggers the apoptotic signaling cascade. While GCV is not recognized by human thymidine kinase, it is recognized as a substrate by some viral thymidine kinases, such as herpes simplex virus-1 thymidine kinase (HSV-TK). As a result, human cells expressing HSV-TK convert GCV to GCV phosphate, which is further phosphorylated and incorporated into synthesized DNA, leading to the termination of synthesis and apoptosis. Examples of HSV-TK mutants include, but are not limited to, TK007 (see Preuss et al., Hum Gene Ther. 2010, Aug;21(8):929-41).

[0094] In some embodiments, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyzes cytosine to uracil, releasing ammonia. Under physiological conditions, the modified site is recognized by an endonuclease, which then breaks the phosphorylation bond in DNA and initiates repair by incorporating a new cytosine. However, cytosine deaminase can also convert 5-fluorocytosine to 5-fluorouracil (5-FU). Therefore, when the non-toxic prodrug 5-FC is provided, cytosine deaminase converts it into the highly toxic 5-FU (a suicide inhibitor of thymidylate synthase), resulting in the inhibition of cell proliferation and apoptosis.

[0095] As used herein, the term "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or enhances the well-being of a subject with respect to the medical treatment of the condition, including, but not limited to, a reduction in the frequency or severity of signs or symptoms of the disease.

[0096] As used herein, the terms "treating" or "treatment" of a disease or condition refers to carrying out a protocol that may include administering one or more therapies to a patient to alleviate signs or symptoms of the disease. In some embodiments, treatment reduces the rate of disease progression, improves or alleviates the disease state, and / or promotes remission or improved prognosis. Alleviation can occur before or after signs or symptoms of the disease or condition appear. Thus, in some embodiments, "treating" or "treatment" includes "preventing" or "prophylaxis" of a disease or 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 provide only a marginal benefit to the patient.

[0097] In various embodiments, a subject in need of treatment may be treated for a disease or for the alleviation of symptoms associated with a disease (e.g., cancer) using a population of modified cells described herein. In some embodiments, the modified cells are immune cells or iPS cells transduced with a CAR or TCR, and further modified to exhibit reduced expression of endogenous MHC class I and MHC class II HLAs genes in combination with the expression or increased expression of certain heterologous genes in the cells, such that when the modified cells are administered to a subject, such cells are less susceptible to killing by the subject's immune system.

[0098] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM", respectively.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. 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 those skilled in the art with a comprehensive dictionary of many of the terms used in this disclosure.

[0100] Units, prefixes, and symbols are shown in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and fractions thereof, where appropriate (e.g., tenths and hundredths of an integer), unless otherwise indicated.

[0101] Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0102] Various aspects described herein are described in further detail in the following subsections.

[0103] II. Compositions of the Present Disclosure Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (eTCR); (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) a polypeptide comprising dominant-negative Fas (Fas-DN), Fas-CD27 chimeric polypeptide (Fas-CD27), Fas-4-1BB chimeric polypeptide (Fas-BB), Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

[0104] Other aspects of the present disclosure are directed to modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) a nucleic acid encoding dominant-negative Fas (Fas-DN); (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; (v) a nucleic acid encoding a suicide gene, or (vi) any combination thereof.

[0105] Other aspects of the present disclosure are directed to modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); and (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0106] Another aspect of the present disclosure is directed to a modified cell comprising (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA), (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA), and (iii) an inactivated endogenous gene encoding CD58.

[0107] Other aspects of the present disclosure are directed to modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; and (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0108] Another aspect of the present disclosure is directed to modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; and (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, and a nucleic acid encoding a suicide gene.

[0109] Other aspects of the present disclosure are directed to dominant-negative Fas (Fas-DN), a Fas-CD27 chimeric polypeptide (Fas-CD27), a Fas-4-1BB chimeric polypeptide (Fas-BB), or a Fas-OX40 chimeric polypeptide (Fas-OX40).

[0110] Another aspect of the present disclosure is directed to a guide RNA capable of hybridizing to the human CD58 gene.

[0111] II.A. Modified Cells Some embodiments of the present disclosure are directed to engineered cells comprising: (i) a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (eTCR); (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) a polypeptide comprising dominant-negative Fas (Fas-DN), Fas-CD27 chimeric polypeptide (Fas-CD27), Fas-4-1BB chimeric polypeptide (Fas-BB), Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

[0112] In some embodiments, the cells disclosed herein have increased in vivo persistence compared to wild-type cells of the same cell type. In some embodiments, the cells disclosed herein are less immunogenic when administered to a human subject. In some embodiments, the human subject mounts a reduced immune response to the cells after administration. In some embodiments, the human subject exhibits reduced NK cell killing of the cells after administration. In some embodiments, NK cell-mediated killing of the cells after 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% compared to wild-type cells of the same cell type administered to a human subject.

[0113] In some embodiments, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In some embodiments, expression of the MHC-I human leukocyte antigens 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% of the expression of the MHC-I human leukocyte antigens in wild-type cells of the same cell type. In some embodiments, the cells have no detectable expression of the MHC-I human leukocyte antigens.

[0114] In some embodiments, the reduced expression of endogenous MHC-I human leukocyte antigens is due to mutation or deletion of one or more endogenous genes encoding beta-2-microglobulin (B2M). In some embodiments, expression of 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% of the expression of B2M in wild-type cells of the same cell type. In some embodiments, the cells have no detectable expression of B2M.

[0115] In some embodiments, the MHC-II human leukocyte antigen is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, expression of the MHC-II human leukocyte antigen 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% of the expression of the MHC-II human leukocyte antigen in wild-type cells of the same cell type. In some embodiments, the cells have no detectable expression of the MHC-II human leukocyte antigen.

[0116] In some embodiments, the reduced expression of endogenous MHC-II human leukocyte antigen is due to mutation or deletion of one or more endogenous genes encoding class II major histocompatibility complex transactivator (CIITA). In some embodiments, the expression of 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% of the expression of CIITA in wild-type cells of the same cell type. In some embodiments, the cells have no detectable expression of CIITA.

[0117] In some embodiments, the cells further comprise reduced expression of endogenous CD58 compared to wild-type cells of the same cell type. In some embodiments, the reduced expression of CD58 is due to mutation or deletion of one or more endogenous genes encoding CD58. In some embodiments, the expression of 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% of the expression of CD58 in wild-type cells of the same cell type. In some embodiments, the cells have no detectable expression of CD58.

[0118] II.A.1. Expression of Fas Constructs In some embodiments, the cell further comprises a nucleic acid encoding Fas-DN, e.g., a Fas-DN disclosed herein. Accordingly, some embodiments of the present disclosure are directed to modified cells, e.g., immune cells, comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-DN. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M; (iii) reduced expression of endogenous CIITA; and (iv) Fas-DN. In some embodiments, the modified cell, e.g., immune cell, comprises (i) a CAR or eTCR; (ii) endogenous reduced expression of B2M; (iii) endogenous reduced expression of CIITA; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-DN.

[0119] In some embodiments, Fas-DN 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: 35, 36, or 40. In some embodiments, Fas-DN is encoded by the nucleotide sequence set forth in SEQ ID NO: 35, 36, or 40.

[0120] [Table 3-1]

[0121] [Table 3-2]

[0122] [Table 3-3]

[0123]

Table 3-4

[0124]

Table 3-5

[0125]

Table 3-6

[0126]

Table 3-7

[0127]

Table 3-8

[0128]

Table 3-9

[0129]

Table 3-10

[0130]

Table 3-11

[0131] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-IHLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-IIHLA compared to wild-type cells of the same cell type; and (iv) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-DN.

[0132] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA relative to wild-type cells of the same cell type; and (iv) Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; and (iv) Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; and (v) Fas-DN.

[0133] In some embodiments, the cells further comprise Fas-CD27, e.g., a Fas-CD27 disclosed herein. Accordingly, some embodiments of the present disclosure are directed to modified cells, e.g., immune cells, comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-CD27.

[0134] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of CIITA; and (iv) a nucleic acid encoding Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of B2M; (iii) reduced expression of CIITA; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) a nucleic acid encoding Fas-CD27.

[0135] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-CD27.

[0136] In some embodiments, the cells further comprise Fas-4-1BB, e.g., Fas-4-1BB as disclosed herein. Accordingly, some embodiments of the present disclosure are directed to modified cells, e.g., immune cells, comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-4-1BB.

[0137] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-4-1BB.

[0138] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; and (v) Fas-4-1BB.

[0139] In some embodiments, the cells further comprise Fas-OX40, e.g., Fas-OX40 as disclosed herein. Accordingly, some embodiments of the present disclosure are directed to modified cells, e.g., immune cells, comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cells, e.g., immune cells, comprise: (i) a CAR or eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-OX40.

[0140] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-OX40.

[0141] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; and (v) Fas-OX40.

[0142] II.A.2. Altered Expression of the Endogenous Poliovirus Receptor (PVR) In some embodiments, the modified cells further comprise reduced expression of endogenous poliovirus receptor (PVR) compared to wild-type cells of the same cell type. In some embodiments, the reduced PVR expression is due to mutation or deletion of one or more endogenous genes encoding PVR. In some embodiments, the expression of PVR 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% of the expression of PVR in wild-type cells of the same cell type. In some embodiments, the cells do not have detectable expression of functional PVR. In some embodiments, the cells have no detectable expression of PVR.

[0143] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-DN.

[0144] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-DN.

[0145] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-CD27.

[0146] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-CD27.

[0147] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-4-1BB.

[0148] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-4-1BB.

[0149] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (v) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; and (vi) Fas-OX40.

[0150] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA relative to wild-type cells of the same cell type; (iv) reduced expression of PVR; and a nucleic acid encoding Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of B2M; (iii) reduced expression of CIITA; (iv) reduced expression of PVR; and (v) a nucleic acid encoding Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of B2M; (iii) reduced expression of CIITA; (iv) reduced expression of CD58; (v) reduced expression of PVR; and (vi) a nucleic acid encoding Fas-OX40.

[0151] II.A.3. Altered Expression of HLA-E In some embodiments, the modified cells further comprise increased expression of HLA-E compared to a wild-type cell of the same cell type, e.g., the cell before modification. In some embodiments, the increased expression of HLA-E results from transfection of a nucleic acid encoding an HLA-E polypeptide. In some embodiments, the increased expression of HLA-E results from modification of an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases expression of the endogenous HLA-E polypeptide. In some embodiments, the increased expression of HLA-E results from (i) transfection of a nucleic acid encoding an HLA-E polypeptide, and (ii) modification of an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases expression of the endogenous HLA-E polypeptide. In some embodiments, the expression of 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%, 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 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%, or at least about 1000% of the expression of HLA-E in a wild-type cell of the same cell type.

[0152] In some embodiments, the modified cells further comprise HLA-E. In some embodiments, expression of 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%, or at least about 100% of the expression of HLA-E in a wild-type cell of the same cell type.

[0153] In some embodiments, the HLA-E is a human HLA-E polypeptide. In some embodiments, the HLA-E polypeptide is a chimeric polypeptide comprising a human HLA-E polypeptide or a portion thereof. In some embodiments, the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a human B2M polypeptide. In some embodiments, 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 set forth in SEQ ID NO: 32.

[0154] In some embodiments, 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: 33, 36, or 38. In some embodiments, the HLA-E is encoded by the nucleotide sequence set forth in SEQ ID NO: 33, 36, or 38.

[0155] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-DN. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (vi) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vii) Fas-DN.

[0156] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (v) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vi) a nucleic acid encoding Fas-DN. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (vi) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vii) Fas-DN.

[0157] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-CD27. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (vi) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vii) Fas-CD27.

[0158] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (v) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vi) Fas-CD27. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (vi) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vii) Fas-CD27.

[0159] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-4-1BB. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (vi) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vii) a nucleic acid encoding Fas-4-1BB.

[0160] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (v) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vi) Fas-4-1BB. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (vi) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vii) Fas-4-1BB.

[0161] In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-OX40. In some embodiments, the modified cells comprise (i) a CAR; (ii) reduced expression of endogenous B2M compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 compared to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (vi) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vii) Fas-OX40.

[0162] In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) increased expression of HLA-E compared to wild-type cells of the same cell type; and (v) Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous MHC-I HLA compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II HLA compared to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR compared to wild-type cells of the same cell type; (v) increased expression of HLA-E compared to wild-type cells of the same cell type; and (vi) Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (v) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vi) Fas-OX40. In some embodiments, the modified cells comprise (i) an eTCR; (ii) reduced expression of endogenous B2M relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous CIITA relative to wild-type cells of the same cell type; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; (v) reduced expression of endogenous PVR relative to wild-type cells of the same cell type; (vi) increased expression of HLA-E relative to wild-type cells of the same cell type; and (vii) Fas-OX40.

[0163] II.A.4. Additional Polypeptides In some embodiments, the engineered cells further overexpress one or more additional endogenous polypeptides. In some embodiments, the engineered cells further express one or more additional heterologous polypeptides. In some embodiments, the engineered cells comprise a human interleukin-15 (IL15) polypeptide. In some embodiments, the engineered cells overexpress an endogenous IL15 polypeptide. In some embodiments, the engineered cells express a heterologous IL15 polypeptide. In some embodiments, the IL15 polypeptide is a membrane-bound IL15 polypeptide. In some embodiments, the IL15 polypeptide is a membrane-bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra). In some embodiments, the IL15 polypeptide comprises an IL15 sushi domain / IL15Ra fusion polypeptide (sushi15). In some embodiments, the IL15 polypeptide comprises a membrane-bound IL15 / IL15Ra-LSP fusion (mIL15 / Ra-LSP). In some embodiments, the IL15 polypeptide comprises a short IL15 polypeptide (sIL15). In some embodiments, the IL15 polypeptide comprises a soluble IL15 polypeptide.

[0164] In some embodiments, the engineered cells comprise a human chemokine (C-C motif) ligand 19 (CCL19) polypeptide. In some embodiments, the engineered cells overexpress an endogenous CCL19 polypeptide. In some embodiments, the engineered cells express a heterologous CCL19 polypeptide.

[0165] In some embodiments, the modified cells comprise an HSVTK polypeptide. In some embodiments, the HSVTK polypeptide 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: 34, 36, or 39. In some embodiments, the HSVTK polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 34, 36, or 39.

[0166] II.A.5.Cells Any cell type can be used in the compositions and methods disclosed herein. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is an embryonic stem cell (ESC). In some embodiments, the cell is an immune cell selected from a T cell, an NK cell, an NKT cell, or a tumor-infiltrating lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is an alpha-beta T cell or a gamma-delta T cell. In some embodiments, the cell is a gamma delta T cell comprising V delta 1. In some embodiments, the cell is a gamma delta T cell comprising V delta 2.

[0167] In some embodiments, the cell is a cell differentiated from iPSC. In some embodiments, the cell is an immune cell differentiated from iPSC. In some embodiments, the cell is a T cell differentiated from iPSC. In some embodiments, the cell is a NK cell differentiated from iPSC. In some embodiments, the cell is a NKT cell differentiated from iPSC.

[0168] In some embodiments, the cells comprise a CAR or eTCR, wherein the CAR or eTCR comprises an antigen binding domain that specifically binds to a tumor antigen. In some embodiments, the tumor antigen is CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha-fetoprotein, 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 gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain. , lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE2, claudins (e.g., CLDN18.2), B7H3, or a combination thereof.

[0169] In some embodiments, the cells comprise a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to mesothelin. In some embodiments, the cells comprise a TCR, wherein the TCR comprises an antigen-binding domain that specifically binds to mesothelin. In some embodiments, the cells comprise an eTCR, wherein the eTCR is a gamma-delta TCR. In some embodiments, the cells comprise an eTCR, wherein the eTCR is a g9d2 TCR.

[0170] In some embodiments, the cells comprise a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to CD 19. In some embodiments, the cells comprise a TCR, wherein the TCR comprises an antigen-binding domain that specifically binds to CD19.

[0171] In some embodiments, the cells comprise a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to BCMA. In some embodiments, the cells comprise a TCR, wherein the TCR comprises an antigen binding domain that specifically binds to BCMA.

[0172] Some embodiments of the present disclosure are directed to a population of cells comprising one or more modified cells disclosed herein. In some embodiments, 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 cells comprises a modification or combination of modifications 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 the cells of the population of cells are modified cells as described herein. In some embodiments, at least about 25% of the population of cells comprise modified cells as disclosed herein. In some embodiments, at least about 30% of the population of cells comprise modified cells as disclosed herein. In some embodiments, at least about 35% of the population of cells comprise modified cells as disclosed herein. In some embodiments, at least about 40% of the population of cells comprise modified cells as disclosed herein. In some embodiments, at least about 45% of the population of cells comprise modified cells disclosed herein. In some embodiments, at least about 50% of the population of cells comprise modified cells disclosed herein. In some embodiments, at least about 55% of the population of cells comprise modified cells disclosed herein. In some embodiments, at least about 60% of the population of cells comprise modified cells disclosed herein.In some embodiments, at least about 65% of the population of cells comprise modified cells disclosed herein. In some embodiments, at least about 70% of the population of cells comprise modified cells disclosed herein. In some embodiments, at least about 75% of the population of cells comprise modified cells disclosed herein.

[0173] II.B. Fas Constructs Some embodiments of the present disclosure are directed to modified and chimeric polypeptides comprising the extracellular domain of the Fas receptor. Further embodiments are directed to engineered cells, e.g., immune cells, e.g., iCAR-T cells, comprising modified or chimeric polypeptides comprising the Fas receptor extracellular domain (FasECD).

[0174] II.B.1.Fas-DN In some embodiments, the modified polypeptide is dominant-negative Fas (Fas-DN). Accordingly, some embodiments of the present disclosure are directed to engineered cells, e.g., immune cells, e.g., iCAR-T cells, comprising Fas-DN. In some embodiments, Fas-DN comprises the extracellular domain of the Fas receptor and one or more amino acid modifications (e.g., deletions or substitutions) within the intracellular (cytoplasmic) domain of the Fas receptor. In some embodiments, Fas-DN comprises the extracellular domain of the Fas receptor and one or more amino acid deletions in the intracellular domain of the Fas receptor. In some embodiments, the modifications (e.g., deletions or substitutions) are in one or more amino acids selected from residues 191-335 of the Fas sequence. In some embodiments, Fas-DN has reduced or absent interaction with Fas-ligand. In some embodiments, Fas-DN is unable to recruit and / or activate caspase-8.

[0175] In some embodiments, Fas-DN comprises the extracellular domain of a Fas receptor and a truncated intracellular domain of a Fas receptor. In some embodiments, the truncated Fas receptor comprises a deletion of one or more amino acids selected from residues 230-312 of the standard Fas receptor amino acid sequence (SEQ ID NO: 30). In some embodiments, the truncated Fas receptor comprises a deletion of 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, or at least about 90 amino acids from residues 230-312 of the standard Fas receptor amino acid sequence (SEQ ID NO: 30). In some embodiments, the truncated Fas receptor comprises a deletion of residues 230-305, residues 230-306, residues 230-307, residues 230-308, residues 230-309, residues 230-310, residues 230-311, residues 230-312, residues 231-312, residues 232-312, residues 233-312, residues 234-312, residues 235-312, residues 236-312, residues 237-312, residues 238-312, residues 239-312, or residues 240-312 of the standard Fas receptor amino acid sequence (SEQ ID NO: 30). In some embodiments, the truncated Fas receptor comprises a deletion of residues 230 to 312 of the canonical Fas receptor amino acid sequence (SEQ ID NO:30) and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 additional amino acids. In some embodiments, the truncated Fas receptor comprises a deletion of residues 230-313, residues 230-314, residues 230-315, residues 230-316, residues 230-317, residues 229-312, residues 228-312, residues 227-312, residues 226-312, or residues 225-312 of the canonical Fas receptor amino acid sequence (SEQ ID NO:30). In some embodiments, the truncated Fas receptor comprises a deletion of residues 230-312 of the canonical Fas receptor amino acid sequence (SEQ ID NO:30).

[0176] In some embodiments, Fas-DN 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 set forth in SEQ ID NO:22. In some embodiments, the chimeric polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, Fas-DN further comprises a signal peptide. In some embodiments, Fas-DN comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, Fas-DN comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO:29.

[0177] II.B.2.Fas-CD27 In some embodiments, the chimeric polypeptide is a Fas-CD27 chimeric polypeptide (Fas-CD27). Accordingly, some embodiments of the present disclosure are directed to engineered cells, e.g., immune cells, e.g., iCAR-T cells, comprising Fas-CD27. In some embodiments, the chimeric polypeptide comprises a Fas extracellular domain linked to the intracellular domain of CD27 or a portion thereof. In some embodiments, the chimeric polypeptide comprises a Fas extracellular domain linked to a portion of the intracellular domain of CD27, wherein the portion of the intracellular domain of CD27 retains one or more intracellular signaling functions of full-length CD27.

[0178] In some embodiments, a Fas-CD27 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 set forth in SEQ ID NO: 22. In some embodiments, a Fas-CD27 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, Fas-CD27 further comprises a signal peptide. In some embodiments, Fas-CD27 comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, Fas-CD27 comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 29. II.B.3.Fas-4-1BB

[0179] In some embodiments, the chimeric polypeptide is a Fas-4-1BB chimeric polypeptide (Fas-4-1BB). Accordingly, some embodiments of the present disclosure are directed to engineered cells, e.g., immune cells, e.g., iCAR-T cells, comprising Fas-4-1BB. In some embodiments, the chimeric polypeptide comprises a Fas extracellular domain linked to the intracellular domain of 4-1BB or a portion thereof. In some embodiments, the chimeric polypeptide comprises a Fas extracellular domain linked to a portion of the intracellular domain of 4-1BB, wherein the portion of the intracellular domain of 4-1BB retains one or more intracellular signaling functions of full-length 4-1BB.

[0180] In some embodiments, a Fas-4-1BB 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 set forth in SEQ ID NO: 23. In some embodiments, a Fas-4-1BB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, Fas-4-1BB further comprises a signal peptide. In some embodiments, Fas-4-1BB comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, Fas-4-1BB comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0181] II.B.4.Fas-OX40 In some embodiments, the chimeric polypeptide is a Fas-OX40 chimeric polypeptide (Fas-OX40). Accordingly, some embodiments of the present disclosure are directed to engineered cells, e.g., immune cells, e.g., iCAR-T cells, comprising Fas-OX40. In some embodiments, the chimeric polypeptide comprises a Fas extracellular domain linked to an intracellular domain of OX40 or a portion thereof. In some embodiments, the chimeric polypeptide comprises a Fas extracellular domain linked to a portion of the intracellular domain of OX40, wherein the portion of the intracellular domain of OX40 retains one or more intracellular signaling functions of full-length OX40.

[0182] In some embodiments, the Fas-OX40 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 set forth in SEQ ID NO: 24. In some embodiments, the chimeric polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24.

[0183] In some embodiments, the Fas-OX40 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 set forth in SEQ ID NO: 25. In some embodiments, the chimeric polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25.

[0184] In some embodiments, the Fas-OX40 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 set forth in SEQ ID NO: 26. In some embodiments, the chimeric polypeptide comprises the amino acid sequence set forth in SEQ ID NO:26.

[0185] In some embodiments, Fas-OX40 further comprises a signal peptide. In some embodiments, Fas-OX40 comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, Fas-OX40 comprises a signal peptide comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0186] II.C. CD58-specific guide RNA Some embodiments of the present disclosure are directed to a guide RNA that can hybridize to the human CD58 gene. In some embodiments, the guide RNA hybridizes to a nucleotide sequence in exon 3 of the endogenous CD58 gene. In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 1-20 and 41.

[0187] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0188] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 2.

[0189] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 3.

[0190] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 4.

[0191] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 5.

[0192] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 6.

[0193] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 7.

[0194] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 8.

[0195] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 9.

[0196] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 10.

[0197] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 11.

[0198] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 12.

[0199] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 13.

[0200] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 14. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 14. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 14.

[0201] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 15.

[0202] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 16.

[0203] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 17.

[0204] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 18.

[0205] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with the guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 19.

[0206] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 20.

[0207] In some embodiments, the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 41. In some embodiments, the guide RNA consists of the nucleotide sequence set forth in SEQ ID NO: 41. In some embodiments, the guide RNA comprises a nucleotide sequence that hybridizes to a nucleotide sequence that overlaps with a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 41.

[0208] Some embodiments of the present disclosure are directed to kits comprising (i) a guide RNA capable of hybridizing to the human CD58 gene disclosed herein, and (ii) a gene editing tool. In some embodiments, the gene editing tool comprises CRISPR / Cas9, CRISPR / Cas12, TALEN, zinc finger endonuclease, or any combination thereof. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:1, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:2, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:3, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:4, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:5, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:6, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:7, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:8, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:9, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:10, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO:11, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 12, and (ii) CRISPR / Cas9.In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 13, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 14, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 15, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 19, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 17, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 18, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 19, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 20, and (ii) CRISPR / Cas9. In some embodiments, the kit comprises (i) a guide RNA comprising the nucleotide sequence set forth in SEQ ID NO: 41, and (ii) CRISPR / Cas9.

[0209] III. Methods of the Disclosure III.A. How to operate Some embodiments of the present disclosure are directed to methods of engineering human cells comprising: (i) transfecting a cell with a nucleic acid encoding a CAR or eTCR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigen, wherein the mutations or deletions result in decreased expression of the MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding MHC-II human leukocyte antigen, wherein the mutations or deletions result in decreased expression of the MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting the cell with a nucleic acid encoding a polypeptide comprising Fas-DN, a Fas-CD27 chimeric polypeptide, a Fas-4-1BB chimeric polypeptide, a Fas-OX40 chimeric polypeptide, or any combination thereof.

[0210] Another aspect of the present disclosure is directed to a method of engineering a human cell comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in decreased expression of MHC-I human leukocyte antigens compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigens, wherein the inactivation results in decreased expression of MHC-II human leukocyte antigens compared to wild-type cells of the same cell type; and (iii) transfecting the cell with a nucleic acid encoding dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0211] Another aspect of the present disclosure is directed to a method of engineering a human cell comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigen, wherein the inactivation results in decreased expression of MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigen, wherein the inactivation results in decreased expression of MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iii) inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in decreased expression of CD58 compared to wild-type cells of the same cell type.

[0212] Another aspect of the present disclosure is directed to a method of engineering a human cell comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigen, wherein the inactivation results in decreased expression of MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigen, wherein the inactivation results in decreased expression of MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; (iii) inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in decreased expression of CD58 compared to wild-type cells of the same cell type; and (iv) transfecting the cell with a nucleic acid encoding dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

[0213] In some embodiments, the CAR or eTCR comprises an antigen binding domain that specifically binds to a tumor antigen, ie, CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha-fetoprotein, 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 gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain. , lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 combination, HER1-HER2 combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen recognized by T cells 1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE, claudins (e.g., CLDN18.2), B7H3, or a combination thereof.

[0214] In some embodiments of the present disclosure, a method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutations or deletions result in decreased expression of the MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutations or deletions result in decreased expression of the MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising Fas-DN, a Fas-CD27 chimeric polypeptide, a Fas-4-1BB chimeric polypeptide, a Fas-OX40 chimeric polypeptide, or any combination thereof.

[0215] In some embodiments of the present disclosure, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising Fas-DN, a Fas-CD27 chimeric polypeptide, a Fas-4-1BB chimeric polypeptide, a Fas-OX40 chimeric polypeptide, or any combination thereof.

[0216] In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-I human leukocyte antigen compared to a wild-type cell of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-II human leukocyte antigen compared to a wild-type cell of the same cell type; and (iv) transfecting the cell with a nucleic acid encoding a polypeptide comprising Fas-DN. In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutation or deletion results in decreased expression of the MHC-I human leukocyte antigen compared to a wild-type cell of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutation or deletion results in decreased expression of the MHC-II human leukocyte antigen compared to a wild-type cell of the same cell type; and (iv) transfecting the cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-CD27 chimeric polypeptide disclosed herein. In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-I human leukocyte antigen compared to a wild-type cell of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-II human leukocyte antigen compared to a wild-type cell of the same cell type; and (iv) transfecting the cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-4-1BB chimeric polypeptide disclosed herein.In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutation or deletion results in decreased expression of the MHC-I human leukocyte antigen compared to a wild-type cell of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutation or deletion results in decreased expression of the MHC-II human leukocyte antigen compared to a wild-type cell of the same cell type; and (iv) transfecting the cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-OX40 chimeric polypeptide disclosed herein.

[0217] In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutations or deletions result in reduced expression of MHC-I human leukocyte antigens compared to wild-type cells of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding MHC-II human leukocyte antigens, wherein the mutations or deletions result in reduced expression of MHC-II human leukocyte antigens compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising Fas-DN. In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-CD27 chimeric polypeptide disclosed herein. In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-I human leukocyte antigen compared to wild-type cells of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutations or deletions result in reduced expression of the MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-4-1BB chimeric polypeptide disclosed herein.In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding an MHC-I human leukocyte antigen, wherein the mutation or deletion results in reduced expression of the MHC-I human leukocyte antigen compared to a wild-type cell of the same cell type; (iii) mutating or deleting one or more endogenous genes encoding an MHC-II human leukocyte antigen, wherein the mutation or deletion results in reduced expression of the MHC-II human leukocyte antigen compared to a wild-type cell of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-OX40 chimeric polypeptide disclosed herein.

[0218] In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding B2M, wherein the mutation or deletion results in reduced expression of B2M compared to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion results in reduced expression of MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising Fas-DN.

[0219] In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding B2M, wherein the mutation or deletion results in reduced expression of B2M compared to a wild-type cell of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion results in reduced expression of MHC-II human leukocyte antigen compared to a wild-type cell of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-CD27 chimeric polypeptide disclosed herein.

[0220] In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding B2M, wherein the mutation or deletion results in reduced expression of B2M compared to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion results in reduced expression of MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-4-1BB chimeric polypeptide disclosed herein.

[0221] In some embodiments, the method comprises: (i) transfecting a cell with a nucleic acid encoding an eTCR; (ii) mutating or deleting one or more endogenous genes encoding B2M, wherein the mutation or deletion results in reduced expression of B2M compared to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion results in reduced expression of MHC-II human leukocyte antigen compared to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide, including, for example, a Fas-OX40 chimeric polypeptide disclosed herein.

[0222] In some embodiments, the method further comprises mutating or deleting one or more endogenous genes encoding CD58, wherein the mutation or deletion results in reduced expression of CD58 compared to a wild-type cell of the same cell type. In some embodiments, mutating or deleting one or more endogenous genes encoding CD58 comprises modifying the CD58 gene using a gene editing tool. In some embodiments, the gene editing tool comprises CRISPR / Cas9, CRISPR / Cas12, TALEN, zinc finger endonuclease, or any combination thereof. In some embodiments, the one or more guide sequences hybridize to a nucleotide sequence of exon 3 of the endogenous CD58 gene. In some embodiments, the one or more guide sequences comprise a nucleotide sequence selected from SEQ ID NOs: 1-20 and 41. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:5. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:8. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:8.In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:9. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:9. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:10. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:10. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:11. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:11. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:12. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:12. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:13. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:13. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:14. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:14. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:15. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO:15. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO:16. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the guide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the guide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the guide sequence consists of ...41.

[0223] In some embodiments, the method further comprises mutating or deleting one or more endogenous genes encoding PVR, wherein the mutation or deletion results in reduced expression of PVR compared to a wild-type cell of the same cell type. Mutating or deleting one or more endogenous genes encoding PVR can be achieved using any method, including, but not limited to, the use of gene editing tools, for example, the gene editing tools disclosed herein.

[0224] In some embodiments, the method further comprises transfecting the cell with a nucleic acid encoding an HLA-E polypeptide, e.g., an HLA-E polypeptide disclosed herein. In some embodiments, the method further comprises modifying an endogenous gene encoding the HLA-E polypeptide, wherein the modification increases expression of the endogenous HLA-E polypeptide. In some embodiments, the method further comprises (i) transfecting the cell with a nucleic acid encoding the HLA-E polypeptide, and (ii) modifying an endogenous gene encoding the HLA-E polypeptide, wherein the modification increases expression of the endogenous HLA-E polypeptide. In some embodiments, the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a B2M polypeptide.

[0225] In some embodiments, the method further comprises transfecting the cell with a nucleic acid encoding an IL15 polypeptide, e.g., an IL15 polypeptide disclosed herein. In some embodiments, the IL15 polypeptide is a membrane-bound IL15 polypeptide. In some embodiments, the IL15 polypeptide is a membrane-bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra). In some embodiments, the IL15 polypeptide comprises an IL15 sushi domain / IL15Ra fusion polypeptide (sushi15). In some embodiments, the IL15 polypeptide comprises a membrane-bound IL15 / IL15Ra-LSP fusion (mIL15 / Ra-LSP). In some embodiments, the IL15 polypeptide comprises a short IL15 polypeptide (sIL15). In some embodiments, the IL15 polypeptide comprises a soluble IL15 polypeptide.

[0226] In some embodiments, the method further comprises transfecting the cell with a nucleic acid encoding a CCL19 polypeptide.

[0227] In some embodiments, an endogenous gene can be inactivated by directing an exogenous gene to the site of the endogenous gene for insertion. In some embodiments, the exogenous gene comprises a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, a nucleic acid encoding a promoter, a nucleic acid encoding a tag protein, or any combination thereof.

[0228] In some embodiments, the promoter is a CAG promoter, an EF1 alpha promoter, a CMV promoter, an hPGK promoter, or any combination thereof. In some embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO:37.

[0229] In some embodiments, the tagged protein is LNGFR or a truncated LNGFR (e.g., delta LNGFR). In some embodiments, the tagged protein is EGFR. In some embodiments, the tagged protein is truncated EGFR (tEGFR).

[0230] In some embodiments, the exogenous gene inserted at the site of the endogenous gene comprises the HLA-E and CAG promoters. In some embodiments, the exogenous gene inserted at the site of the endogenous gene comprises the FasDN and CAG promoters. In some embodiments, the exogenous gene inserted at the site of the endogenous gene comprises the HLA-E, HSV-TK, FasDN, and CAG promoters. In some embodiments, the exogenous gene inserted at the site of the endogenous gene comprises the HLA-E, HSV-TK, FasDN, EF1 alpha promoter, and CAG promoter. In some embodiments, the exogenous gene inserted at the site of the endogenous gene comprises the HLA-E, HSV-TK, FasDN, LNGFR, and CAG promoters.

[0231] In some embodiments, the exogenous gene is inserted at the site of the B2M gene. In some embodiments, the exogenous gene is inserted at the site of the CIITA gene. In some embodiments, the exogenous gene is inserted at the site of the CD58 gene.

[0232] In some embodiments, the exogenous gene is a nucleic acid comprising a nucleic acid sequence selected from SEQ ID NOs: 33-40.

[0233] In some embodiments, the cells are iPSCs. In some embodiments, the cells are cells differentiated from iPSCs. In some embodiments, the cells are immune cells differentiated from iPSCs. In some embodiments, the cells are T cells differentiated from iPSCs. In some embodiments, the cells are NK cells differentiated from iPSCs. In some embodiments, the cells are NKT cells differentiated from iPSCs. When the cells are iPSCs, they can be used as a source of not only immune cells but also any other type of cells that can be differentiated from iPSCs. Such differentiated cells, which have reduced immunogenicity in vivo, are useful for any cell therapy that involves administering or transplanting such cells into a patient.

[0234] III.B. Treatment Methods Some embodiments of the present disclosure are directed to methods of treating a disease or condition in a subject in need of treatment, comprising administering to the subject a composition disclosed herein. In some embodiments, the method comprises administering a modified or engineered cell disclosed herein. In some embodiments, the method comprises administering a population of cells disclosed herein. In some embodiments, the method comprises administering a composition comprising a Fas construct disclosed herein. In some embodiments, the method comprises administering a composition comprising a CD58-specific guide RNA.

[0235] In some embodiments, the disease or condition includes cancer, e.g., the subject is afflicted with cancer. In some embodiments, the cancer is selected from the group consisting of bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like. The cancer may be a leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory or recurrent after one or more prior anti-cancer therapies. In some embodiments, the one or more prior anti-cancer therapies comprise standard of care.

[0236] In some embodiments, the compositions disclosed herein are administered in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy comprises chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof. In some embodiments, the additional anti-cancer therapy comprises chemotherapy. In some embodiments, the additional anti-cancer therapy comprises an immune checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy comprises a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR antagonist, or any combination thereof. In some embodiments, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-1. In some embodiments, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-L1.

[0237] In some embodiments, the method further comprises pretreating the subject prior to administering the population of immune cells. In some embodiments, the subject is administered chemotherapy prior to administering the population of immune cells. In some embodiments, the subject is administered immunodepleting chemotherapy prior to administering the population of immune cells. In some embodiments, the immunodepleting chemotherapy comprises cyclophosphamide, fludarabine, or both.

[0238] In some embodiments, the method comprises administering to the subject (i) the expanded population of cells and (ii) a cytokine, hi some embodiments, the cytokine comprises IL-2, an analog thereof, a variant thereof, or a fragment thereof.

[0239] In some embodiments, the cells of the present disclosure are administered to a patient in need thereof at least about 1 x 10 6 Cells, at least approximately 2 x 10 6 cells, at least approximately 3 x 10 6 Cells, at least approximately 4 x 10 6 cells, at least approximately 5 x 10 6 cells, 1 x 10 7 Cells, at least approximately 2 x 10 7 cells, at least approximately 3 x 10 7 Cells, at least approximately 4 x 10 7 cells, at least approximately 5 x 10 7 cells, 1 x 10 8 Cells, at least approximately 2 x 10 8 cells, at least approximately 3 x 10 8 Cells, at least approximately 4 x 10 8 cells, at least approximately 5 x 10 8 cells, 1 x 10 9 Cells, at least approximately 2 x 10 9 cells, at least approximately 3 x 10 9 Cells, at least approximately 4 x 10 9 cells, or at least about 5 x 10 9 The cells are administered to the subject in a dose of 0.5 mg / kg of the antibody.

[0240] III.C. Cell Therapy Formulations Some aspects of the present disclosure are directed to a cell population comprising a plurality of modified cells disclosed herein. Some aspects of the present disclosure are directed to a cell therapy comprising a population of modified cells disclosed herein. In some aspects, the population of cells is cryopreserved. Any method for cryopreserving cells, e.g., immune cells, can be used in the methods and compositions disclosed herein. In some aspects, the cells are cryopreserved in the presence of DMSO. In some aspects, the cell therapy is cryopreserved to facilitate cell transport.

[0241] The cell therapy agent and / or cell population disclosed herein can be further formulated with one or more excipients. Any excipient that can preserve cells can be used in the methods and compositions disclosed herein. In some embodiments, the cell therapy agent and / or cell population is formulated with one or more excipients that enable cryopreservation of cells, such as DMSO.

[0242] In some embodiments, a population of cells comprises a plurality of modified cells disclosed herein and one or more additional cells. In some embodiments, 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% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 10% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 20% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 25% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 30% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 40% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 50% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 60% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 65% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 70% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 75% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 80% of the cells in the population of cells comprise a plurality of modified cells disclosed herein. In some embodiments, at least about 90% of the cells in the population of cells comprise a plurality of modified cells disclosed herein.

[0243] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD19. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD19. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD19.

[0244] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD20. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD20. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD20.

[0245] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to ROR1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to ROR1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to ROR1.

[0246] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD22. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD22. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD22.

[0247] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a carcinoembryonic antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a carcinoembryonic antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a carcinoembryonic antigen.

[0248] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds alpha-fetoprotein. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds alpha-fetoprotein. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds alpha-fetoprotein.

[0249] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CA-125. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds CA-125. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CA-125.

[0250] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to 5T4. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to 5T4. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to 5T4.

[0251] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MUC-1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MUC-1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MUC-1.

[0252] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to an epithelial tumor antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to an epithelial tumor antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to an epithelial tumor antigen.

[0253] Some aspects of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to prostate-specific antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a prostate-specific antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a prostate-specific antigen.

[0254] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a melanoma-associated antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a melanoma-associated antigen. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a melanoma-associated antigen.

[0255] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to mutated p53. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to mutated p53. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to mutated p53.

[0256] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a mutated ras. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of engineered cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a mutated ras. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of engineered cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a mutated ras.

[0257] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER2 / Neu. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER2 / Neu. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER2 / Neu.

[0258] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to folate-binding protein. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a folate-binding protein. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to folate-binding protein.

[0259] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the HIV-1 envelope glycoprotein gp120. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the HIV-1 envelope glycoprotein gp120. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the HIV-1 envelope glycoprotein gp120.

[0260] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the HIV-1 envelope glycoprotein gp41. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the HIV-1 envelope glycoprotein gp41. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the HIV-1 envelope glycoprotein gp41.

[0261] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GD2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GD2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GD2.

[0262] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD123. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD123. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD123.

[0263] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD33. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD33. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD33.

[0264] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD138. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD138. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD138.

[0265] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD23. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD23. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD23.

[0266] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD30. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD30. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD30.

[0267] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD56. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD56. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to CD56.

[0268] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to c-Met. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to c-Met. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to c-Met.

[0269] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds mesothelin. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds mesothelin. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds mesothelin.

[0270] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GD3. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GD3. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GD3.

[0271] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HERV-K. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HERV-K. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HERV-K.

[0272] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to IL-11Ralpha. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to IL-11Ralpha. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to IL-11Ralpha.

[0273] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the IL-kappa chain. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a kappa chain. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a kappa chain.

[0274] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to the lambda chain. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a lambda chain. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a lambda chain.

[0275] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GSPG4. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GSPG4. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GSPG4.

[0276] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to ERBB2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to ERBB2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to ERBB2.

[0277] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to EGFRvIII. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to EGFRvIII. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to EGFRvIII.

[0278] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to VEGFR2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to VEGFR2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to VEGFR2.

[0279] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER2-HER3 in combination. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER2-HER3 in combination. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of engineered cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising antigen-binding domains that specifically bind to HER2-HER3 in combination.

[0280] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER1-HER2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER1-HER2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to HER1-HER2.

[0281] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to NY-ESO-1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to NY-ESO-1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to NY-ESO-1.

[0282] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to SSX2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to SSX2. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to SSX2.

[0283] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MAGE. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a MAGE. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to a MAGE.

[0284] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MART-1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MART-1. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to MART-1.

[0285] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to gp100. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to gp100. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to gp100.

[0286] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds PSA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds PSA.

[0287] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSMA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSMA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSMA.

[0288] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSCA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSCA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to PSCA.

[0289] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GPC3. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GPC3. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to GPC3.

[0290] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to EpCAM. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to EpCAM. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (v) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds to EpCAM.

[0291] Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type or compared to the cells before modification; (iii) increased expression of Fas (Fas-DN), HLA-E compared to wild-type cells of the same cell type or compared to the cells before modification, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds BCMA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof; and (iv) a chimeric antigen receptor comprising an antigen-binding domain that specifically binds BCMA. Some embodiments of the present disclosure are directed to a population of cells comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding an MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endo...

Claims

1. 1. A modified cell comprising: (i) a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor (eTCR); (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iv) a polypeptide comprising a dominant-negative Fas (Fas-DN), a Fas-CD27 chimeric polypeptide (Fas-CD27), a Fas-4-1BB chimeric polypeptide (Fas-BB), a Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

2. The cell of claim 1, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C.

3. The cell according to claim 1, wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR.

4. The cell of claim 1, wherein the reduced expression of the MHC-I human leukocyte antigen is due to a mutation or deletion of one or more endogenous genes encoding beta-2-microglobulin (B2M).

5. The cell of claim 1, wherein the reduced expression of the endogenous MHC-II human leukocyte antigen is due to a mutation or deletion of one or more endogenous genes encoding class II major histocompatibility complex transactivators (CIITAs).

6. The cell of claim 1, further comprising reduced expression of endogenous CD58 compared to a wild-type cell of the same cell type.

7. The cell of claim 6, wherein the decreased expression of CD58 is due to a mutation or deletion of one or more endogenous genes encoding CD58.

8. The cell according to any one of claims 1 to 7, which comprises a nucleic acid encoding Fas-DN.

9. The cell according to any one of claims 1 to 7, comprising a nucleic acid encoding Fas-CD27.

10. The cell according to any one of claims 1 to 7, comprising a nucleic acid encoding Fas-BB.

11. The cell according to any one of claims 1 to 7, comprising a nucleic acid encoding Fas-OX40.

12. 1. A modified cell comprising: (i) with a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iv) Fas-DN.

13. A cell, (i) with a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iv) Fas-CD27.

14. A cell, (i) with a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iv) Fas-BB.

15. A cell, (i) with a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iv) Fas-OX40.

16. The cell of any one of claims 12 to 15, further comprising reduced expression of endogenous CD58 compared to a wild-type cell of the same cell type.

17. The cell of claim 16, wherein the decreased expression of CD58 is due to a mutation or deletion of one or more endogenous genes encoding CD58.

18. 18. The cell of any one of claims 1 to 17, further comprising reduced expression of endogenous poliovirus receptor (PVR) compared to a wild-type cell of the same cell type.

19. The cell of claim 18, wherein the decreased expression of PVR is due to a mutation or deletion of one or more endogenous genes encoding PVR.

20. The cell of any one of claims 1 to 19, further comprising increased expression of HLA-E compared to a wild-type cell of the same cell type.

21. The cell of claim 20, wherein the increased expression of HLA-E is due to (i) transfection of a nucleic acid encoding an HLA-E polypeptide; (ii) modification of an endogenous gene encoding an HLA-E polypeptide; or (iii) both (i) and (ii), wherein the modification increases the expression of the endogenous HLA-E polypeptide.

22. The cell of claim 21, wherein the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a human B2M polypeptide.

23. The cell of any one of claims 1 to 22, further comprising a human interleukin-15 (IL15) polypeptide.

24. The cell of claim 23 , wherein the human IL15 polypeptide is a membrane-bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra).

25. The cell of any one of claims 1 to 24, further comprising a human chemokine (CC motif) ligand 19 (CCL19) polypeptide.

26. The cell according to any one of claims 1 to 25, wherein the cell is an immune cell, an induced pluripotent stem cell (iPSC), or a cell differentiated from an iPSC.

27. The cell of any one of claims 1 to 26, wherein the cell is (i) an immune cell differentiated from an iPSC, or (ii) a hematopoietic stem cell differentiated from an iPSC.

28. The cell of any one of claims 1 to 27, wherein the cell comprises a T cell, a NK cell, a NKT cell, or a tumor-infiltrating lymphocyte.

29. The cell of any one of claims 1 to 28, wherein the CAR or the eTCR comprises an antigen-binding domain that specifically binds to a tumor antigen.

30. The tumor antigen is selected from the group consisting of CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha-fetoprotein, 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 gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, κ chain, λ chain, C 30. The cell of claim 29, comprising SPG4, 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 1 recognized by T cells (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), GPC3, EpCAM, BCMA, GCC, ADGRE, claudins, B7H3, or a combination thereof.

31. The cell of any one of claims 1 to 30, comprising a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to mesothelin.

32. 1. A method of engineering a human cell, comprising: (i) introducing into said cells a nucleic acid encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR); (ii) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigen (HLA), wherein said inactivation results in reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigens (HLA), wherein said inactivation results in reduced expression of MHC-II human leukocyte antigens (HLA) compared to wild-type cells of the same cell type; (iv) introducing into the cell a nucleic acid encoding a polypeptide comprising a dominant-negative Fas (Fas-DN), a Fas-CD27 chimeric polypeptide (Fas-CD27), a Fas-4-1BB chimeric polypeptide (Fas-BB), a Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

33. moreover 33. The method of claim 32, comprising (v) inactivating one or more endogenous genes encoding CD58, wherein said inactivation results in decreased expression of CD58 compared to wild-type cells of the same cell type.

34. 34. The method of claim 33, wherein said inactivation of said one or more endogenous genes encoding CD58 comprises modifying said CD58 gene using a gene editing tool.

35. 35. The method of Claim 33 or 34, wherein the gene editing tool comprises CRISPR / Cas9, CRISPR / Casl2, a TALEN, a zinc finger endonuclease, or any combination thereof.

36. 36. The method of any one of claims 33 to 35, wherein the inactivation of the one or more endogenous genes encoding CD58 comprises 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 CD58 gene.

37. 37. The method of Claim 36, wherein the one or more guide sequences hybridize to a nucleotide sequence of exon 3 of the endogenous CD58 gene.

38. 38. The method of claim 36 or 37, wherein the one or more guide sequences comprise a nucleotide sequence selected from SEQ ID NOs: 1-20 and 41.

39. 39. The method of any one of claims 32-38, further comprising inactivating one or more endogenous genes encoding poliovirus receptor (PVR), wherein said inactivation results in reduced expression of PVR compared to a wild-type cell of the same cell type.

40. 40. The method of any one of claims 32 to 39, 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 expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii).

41. 41. The method of claim 40, wherein the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a human B2M polypeptide.

42. 42. The method of any one of claims 32 to 41, further comprising transfecting said cells with a nucleic acid encoding an interleukin-15 (IL15) polypeptide.

43. 43. The method of claim 42, wherein the IL15 polypeptide is a membrane-bound IL15 / IL15Ralpha fusion polypeptide (mIL15 / Ra).

44. 44. The method of any one of claims 32 to 43, further comprising introducing into said cells a heterologous nucleic acid encoding a human chemokine (CC motif) ligand 19 (CCL19) polypeptide.

45. The method of any one of claims 32 to 44, wherein the cell is an immune cell, an induced pluripotent stem cell (iPSC), or a cell differentiated from an iPSC.

46. The method of any one of claims 32 to 45, wherein the cells are immune cells differentiated from iPSCs or hematopoietic stem cells.

47. The method of any one of claims 32 to 46, wherein the cells comprise T cells, NK cells, NKT cells, or tumor-infiltrating lymphocytes.

48. The method of any one of claims 32 to 47, wherein the CAR or the eTCR comprises an antigen-binding domain that specifically binds to a tumor antigen.

49. The tumor antigen is selected from the group consisting of CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha-fetoprotein, 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 gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, and κ.

49. The method of claim 48, comprising a HER2-HER3 combination, a HER1-HER2 combination, a HER2-HER3 combination, a HER1-HER2 combination, a NY-ESO-1, a synovial sarcoma X breakpoint 2 (SSX2), a melanoma antigen (MAGE), a melanoma antigen 1 recognized by T cells (MART-1), gp100, a prostate-specific antigen (PSA), a prostate-specific membrane antigen (PSMA), a prostate stem cell antigen (PSCA), GPC3, BCMA, GCC, ADGRE, a claudin, or a combination thereof.

50. A cell prepared according to the method of any one of claims 32 to 49.

51. 51. The cell of any one of claims 1 to 31 and 50, which has increased persistence in vivo compared to a wild-type cell of the same cell type.

52. A population of cells comprising the cells of any one of claims 1 to 31, 50 and 51.

53. A cell population, wherein at least 50% of the cells in the population comprise a cell according to any one of claims 1 to 31, 50 and 51.

54. 54. A method of treating a subject in need thereof, comprising administering to said subject a cell according to any one of claims 1 to 31, 50 and 51 or a population of cells according to claim 52 or 53.

55. 55. The method of claim 54, wherein the subject is suffering from cancer.

56. The cancer may be bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, thyroid cancer ...

56. The method of claim 55, comprising treating cancers including myeloid leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof.

57. A guide RNA capable of hybridizing to a human CD58 gene, said guide RNA comprising a nucleic acid sequence selected from SEQ ID NOs: 1 to 20 and 41.

58. 58. The guide RNA of claim 57, comprising the nucleic acid sequence set forth in SEQ ID NO:

1.

59. 58. The guide RNA of claim 57, consisting of the nucleic acid sequence set forth in SEQ ID NO:

1.

60. 58. The guide RNA of claim 57, comprising the nucleic acid sequence set forth in SEQ ID NO:

2.

61. 58. The guide RNA of claim 57, consisting of the nucleic acid sequence set forth in SEQ ID NO:

2.

62. 58. The guide RNA of claim 57, comprising the nucleic acid sequence set forth in SEQ ID NO:

3.

63. 58. The guide RNA of claim 57, consisting of the nucleic acid sequence set forth in SEQ ID NO:

3.

64. 58. The guide RNA of claim 57, comprising the nucleic acid sequence set forth in SEQ ID NO:

4.

65. 58. The guide RNA of claim 57, consisting of the nucleic acid sequence set forth in SEQ ID NO:

4.

66. 66. A method for inactivating the human CD58 gene in a cell, the method comprising contacting the cell with a guide RNA according to any one of claims 57 to 65, or a nucleic acid encoding said guide RNA, and a DNA endonuclease or a nucleic acid encoding said DNA endonuclease.

67. 60. The method of Claim 59, wherein the DNA endonuclease comprises CRISPR / Cas9.

68. A dominant-negative Fas (Fas-DN) comprising the amino acid sequence set forth in SEQ ID NO:

27.

69. A Fas-CD27 chimeric polypeptide (Fas-CD27) comprising the amino acid sequence set forth in SEQ ID NO:

22.

70. A Fas-4-1BB chimeric polypeptide (Fas-4-1BB) comprising the amino acid sequence set forth in SEQ ID NO:

23.

71. A Fas-OX40 chimeric polypeptide (Fas-OX40) comprising the amino acid sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:

26.

72. The cell of claim 1, wherein the eTCR is a gamma-delta TCR.

73. 73. The cell of claim 72, wherein the gamma-delta TCR is a V gamma 9-V delta 2 TCR (g9d2 TCR).

74. 1. A modified cell comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (iii) the modified cell comprising expression of Fas (Fas-DN), increased expression of HLA-E compared to a wild-type cell of the same cell type, a nucleic acid encoding a suicide gene, or any combination thereof.

75. 1. A modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (i) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) the modified cell, comprising a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

76. 1. A modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (i) an inactivated endogenous gene encoding an MHC-II human leukocyte antigen (HLA); (iii) an inactivated endogenous gene encoding CD58; (iv) the modified cell, comprising a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

77. 1. A method of engineering a human cell, comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigen (HLA), wherein said inactivation results in reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigens (HLA), wherein said inactivation results in reduced expression of MHC-II human leukocyte antigens (HLA) compared to wild-type cells of the same cell type; (iii) transfecting the cells with a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

78. 1. A method of engineering a human cell, comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigen (HLA), wherein said inactivation results in reduced expression of MHC-I human leukocyte antigen (HLA) compared to wild-type cells of the same cell type; (ii) inactivating one or more endogenous genes encoding MHC-II human leukocyte antigens (HLA), wherein said inactivation results in reduced expression of MHC-II human leukocyte antigens (HLA) compared to wild-type cells of the same cell type; (iii) inactivating one or more endogenous genes encoding CD58, wherein said inactivation results in decreased expression of CD58 compared to wild-type cells of the same cell type; (iv) transfecting the cells with a nucleic acid encoding a dominant-negative Fas (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

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