TW202417617A - Hypoimmunogenic modified cells - Google Patents

Abstract

The present disclosure relates to modified cells, e.g., chimeric antigen receptor (CAR) T cells and/or exogenous T cell receptor (eTCR) cells, which have reduced in vivoimmunogenicity, and the use thereof.

Description

Modified cells with low immunogenicity

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

Cell-based immunotherapy is a rapidly evolving area of research used to develop new and improved treatments for a variety of diseases, including cancer. Several therapies have been approved, including isolating T cells from an individual, modifying the T cells to express a chimeric antigen receptor (CAR) or exogenous T cell receptor (eTCR) that enables the T cells to target a specific antigen, and re-administering the modified T cells to the same individual. Although autologous CAR-T or eTCR therapy has proven very promising, the process can be time-consuming and expensive, and has inconsistent results due, at least in part, to donor variability.

One approach to simplifying this process is to create "off-the-shelf" modified immune cells derived from induced pluripotent stem cells (iPSCs). However, administering HLA-mismatched iPSC-derived immune cells to patients will likely generate an immune response in the patient, causing the patient to reject the cell therapy. Therefore, there remains a need in this area to develop iPSC-derived cell therapies that can evade the patient's immune response.

The present disclosure provides, at least in part, novel methods and modified cells that, when administered to a patient as a cell therapy product (e.g., immune cells or induced pluripotent cell populations transduced with CAR or TCR and containing certain modifications described herein), are able to evade killing by the patient's immune system. The present invention is based at least in part on the discovery that reducing the expression of endogenous MHC-I HLA and MHC-II HLA, while expressing dominant negative Fas (Fas-DN) and HLA-E in cells (e.g., immune cells) unexpectedly enables cells to have longer persistence in vivo by not only escaping natural killer (NK) cell-mediated killing and T cell-mediated killing, but also by avoiding Fas-mediated apoptosis. Such a combination of modifications helps to remove a major obstacle to existing immune cell therapies.

Some aspects of the present disclosure relate to 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) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (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.

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

In some aspects, the cell further comprises endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type. In some aspects, the reduced expression of CD58 is caused by a mutation or deletion of one or more endogenous genes encoding CD58. In some aspects, the cell comprises a nucleic acid encoding Fas-DN. In some aspects, the cell comprises a nucleic acid encoding Fas-CD27. In some aspects, the cell comprises a nucleic acid encoding Fas-BB. In some aspects, the cell comprises a nucleic acid encoding Fas-OX40.

Some aspects of the present disclosure relate to a modified cell comprising: (i) a CAR or eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-DN.

Some aspects of the present disclosure relate to a cell comprising: (i) a CAR or an eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-CD27.

Some aspects of the present disclosure relate to a cell comprising: (i) a CAR or an eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-BB.

Some aspects of the present disclosure relate to a cell comprising: (i) a CAR or an eTCR; (ii) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-OX40.

In some aspects, the cell further comprises reduced endogenous CD58 expression relative to wild-type cells of the same cell type. In some aspects, reduced expression of CD58 is caused by a mutation or deletion of one or more endogenous genes encoding CD58.

In some aspects, the cell further comprises an endogenous poliovirus receptor (PVR) that is reduced in expression relative to wild-type cells of the same cell type. In some aspects, the reduced expression of PVR is caused by a mutation or deletion of one or more endogenous genes encoding PVR.

In some aspects, the cell further comprises increased expression of HLA-E relative to wild-type cells of the same cell type. In some aspects, the increased expression of HLA-E is caused by one of: (i) introducing a nucleic acid encoding an HLA-E polypeptide into the cells; and (ii) modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification results in increased expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii). In some aspects, the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a B2M polypeptide.

In some aspects, the method further comprises transfecting the cell with a nucleic acid encoding an interleukin 15 (IL15) polypeptide. In some aspects, the IL15 polypeptide is a membrane-bound IL15/IL15Rα fusion polypeptide (mIL15/Ra).

In some aspects, the method further comprises introducing a heterologous nucleic acid encoding a human interleukin (C-C motif) ligand 19 (CCL19) polypeptide into the cell.

In some embodiments, the cell is an immune cell, an induced pluripotent stem cell (iPSC), or a cell differentiated from an iPSC. In some embodiments, the cell is an immune cell or a hematopoietic stem cell differentiated from an iPSC. In some embodiments, the cell comprises a T cell, a NK cell, a NKT cell, or a tumor infiltrating lymphocyte.

In some aspects, the CAR or eTCR comprises an antigen binding domain that specifically binds to a tumor antigen. In some aspects, the tumor antigen comprises CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-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, BCMA, GCC, ADGRE, claudin, or any combination thereof.

Some aspects of the disclosure relate to cells prepared according to the methods disclosed herein. In some aspects, the cells have increased persistence in vivo relative to wild-type cells of the same cell type.

Some aspects of the disclosure relate to a cell population comprising a cell disclosed herein.

Some aspects of the disclosure relate to a population of cells, wherein at least 50% of the cells in the population comprise cells disclosed herein.

Some aspects of the disclosure relate to a method of treating a subject in need thereof, the method comprising administering to the subject a cell or cell population disclosed herein.

In some embodiments, the subject has cancer. In some embodiments, the cancer comprises bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, 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, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem neurofibromas, pituitary adenomas, Kaposi's sarcoma (Kaposi's sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer including asbestos-induced cancer, or any combination thereof.

Some aspects of the present disclosure relate to a guide RNA capable of hybridizing with a human CD58 gene, comprising a nucleic acid sequence selected from SEQ ID NOs: 1-20 and 41. In some aspects, the guide RNA comprises the nucleic acid sequence shown in SEQ ID NO: 1. In some aspects, the guide RNA consists of the nucleic acid sequence shown in SEQ ID NO: 1. In some aspects, the guide RNA comprises the nucleic acid sequence shown in SEQ ID NO: 2. In some aspects, the guide RNA consists of the nucleic acid sequence shown in SEQ ID NO: 2. In some aspects, the guide RNA comprises the nucleic acid sequence shown in SEQ ID NO: 3. In some aspects, the guide RNA consists of the nucleic acid sequence shown in SEQ ID NO: 3. In some aspects, the guide RNA comprises the nucleic acid sequence shown in SEQ ID NO: 4. In some aspects, the guide RNA consists of the nucleic acid sequence shown in SEQ ID NO: 4.

Some aspects of the disclosure relate to a method of inactivating a human CD58 gene in a cell, the method comprising contacting the cell with a CD58 guide RNA or a nucleic acid encoding the guide RNA disclosed herein and a DNA endonuclease or a nucleic acid encoding the DNA endonuclease. In some aspects, the DNA endonuclease comprises CRISPR/Cas9.

Some aspects of the present disclosure relate to a dominant negative Fas (Fas-DN) comprising the amino acid sequence shown in SEQ ID NO: 27.

Some aspects of the present disclosure relate to a Fas-CD27 chimeric polypeptide (Fas-CD27) comprising the amino acid sequence shown in SEQ ID NO: 22.

Some aspects of the present disclosure relate to a Fas-4-1BB chimeric polypeptide (Fas-4-1BB) comprising the amino acid sequence shown in SEQ ID NO: 23.

Some aspects of the disclosure relate to a Fas-OX40 chimeric polypeptide (Fas-OX40) comprising the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

In some embodiments, the eTCR is a γ-δ TCR. In some embodiments, the γ-δ TCR is a Vγ9-Vδ2 TCR (g9d2TCR).

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

Some aspects of the present disclosure relate to a modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a method of engineering a human cell, the method comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of the MHC-I human leukocyte antigen relative 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 reduced expression of the MHC-II human leukocyte antigen relative to wild-type cells of the same cell type; and (iii) transfecting the cell 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.

Some aspects of the present disclosure relate to a method of engineering human cells, the method comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of the MHC-I human leukocyte antigens relative 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 reduced expression of the MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; (iii) inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in reduced expression of CD58 relative to wild-type cells of the same cell type; and (iv) inactivating one or more endogenous genes encoding a dominant negative Fas The cell is transfected with a nucleic acid encoding a Fas-DN (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

Cross-references to related applications

This application claims the benefit of priority to U.S. Provisional Application No. 63/371,060, filed on August 10, 2022, which is incorporated herein by reference in its entirety. Citation of Sequence Listing Submitted Electronically via EFS-WEB

The contents of the electronically submitted sequence listing submitted 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.

Some aspects of the present disclosure relate to modified 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) relative to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (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.

Some aspects of the present disclosure relate to modified cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-DN.

Some aspects of the present disclosure relate to modified cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-CD27.

Some aspects of the present disclosure relate to modified cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-BB.

Some aspects of the present disclosure relate to modified cells comprising: (i) a CAR or eTCR; (ii) reduced expression of MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (iii) reduced expression of MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv) Fas-OX40.

In some aspects, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In some aspects, the reduced expression of the MHC-I human leukocyte antigens or the inactivation of endogenous genes encoding the MHC-I human leukocyte antigens is caused by mutation, transgene insertion (knock-in), or deletion (knockout) of one or more endogenous genes encoding beta-2-microglobulin (B2M). In some aspects, the reduced expression of the MHC-I human leukocyte antigens or the inactivation of endogenous genes encoding the MHC-I human leukocyte antigens is caused by the insertion (knock-in) of a transgene encoding HLA-E or a protein comprising HLA-E in whole or in part into one or more endogenous genes encoding beta-2-microglobulin (B2M). In some aspects, the transgene encoding HLA-E or a protein comprising HLA-E can be inserted in whole or in part into the site of exon 1, exon 2 or exon 3 of the endogenous gene encoding beta-2-microglobulin (B2M), preferably into the site of exon 1. In some aspects, the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR. In some aspects, the reduced expression of endogenous MHC-II human leukocyte antigens or the inactivation of endogenous genes encoding MHC-II human leukocyte antigens is caused by mutation, transgene insertion or deletion (knockout) of one or more endogenous genes encoding class II major histocompatibility complex transactivator protein (CIITA). In some aspects, the reduced expression of MHC-II human leukocyte antigens or the inactivation of endogenous genes encoding MHC-II human leukocyte antigens is caused by the insertion (knock-in) of a transgene encoding Fas (e.g., Fas-DN), a suicide gene (e.g., HSV-TK or a variant thereof) and/or a tag protein (e.g., EGFR, LNGFR or a variant thereof) in one or more endogenous genes encoding CIITA. In some aspects, the transgene encoding Fas (e.g., Fas-DN), a suicide gene (e.g., HSV-TK or a variant thereof) and/or a tag protein (e.g., EGFR, LNGFR or a variant thereof) can be inserted in whole or in part into the site of exon 1, exon 2 or exon 3 of the endogenous gene encoding CIITA, preferably into the site of exon 3. The transgene may include the whole or part of a gene encoding one or more proteins including Fas (e.g., Fas-DN), a suicide gene (e.g., HSV-TK or a variant thereof), and/or a tag protein (e.g., EGFR, LNGFR or a variant thereof). In some aspects, the reduced expression of CD58 or the inactivation of the endogenous gene encoding CD58 is caused by a mutation, transgene insertion (knock-in) or deletion (knockout) of one or more endogenous genes encoding CD58. The deletion (knockout) site of one or more endogenous genes encoding CD58 may be exon 1, exon 2 and/or exon 3 of the endogenous gene encoding CD58, preferably exon 3.

Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific compositions or method steps described, which may, of course, vary. It should be apparent to those skilled in the art after reading the present disclosure that each of the aspects described and illustrated herein each has separate components and features that can be easily separated or combined with the features of any other several aspects without departing from the scope or spirit of the present disclosure. Any enumerated method may be performed in the order of events enumerated or in any other order that is logically feasible.

The headings provided herein are not limitations of the various aspects of the present disclosure, which can be defined by reference to the entire specification. It should also be understood that the terminology used herein is only used for the purpose of describing specific aspects and is not intended to be limiting. I. Terminology

In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, the following terms shall each have the meanings set forth below. Additional definitions are set forth throughout this application.

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

Throughout this disclosure, the term "a/an" entity refers to one or more of the entity; for example, "a chimeric polypeptide" should be understood to mean one or more chimeric polypeptides. Thus, the terms "a", "one or more", and "at least one" are used interchangeably herein.

In addition, the use of "and/or" herein should be considered as a specific disclosure about each of the two specified features or components with or without the other. Therefore, as used herein in phrases such as "A and/or B", the term "and/or" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, as used in phrases such as "A, B, and/or C", the term "and/or" is intended to cover each of the following: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In addition, the use of "or" also means an open list of components in the list. For example, "wherein X comprises A or B" means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.

The term "about" or "substantially comprising" refers to a value or composition that is within an acceptable error range of the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on the manner in which the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 10%. In addition, particularly for biological systems or processes, the terms may mean up to an order of magnitude or up to 5 times a value. When a particular value or composition is provided in the present application and the claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range of the particular value or composition.

The terms “activated immune cells”, “activated T cells” and “activated NK cells” refer in particular to immune cells undergoing cell division, such as T cells and/or NK cells.

"Antigen" refers to any molecule, such as a peptide, that evokes an immune response or is capable of being bound by a TCR. The immune response may involve the production of antibodies, or the activation of cells with specific immune competence, or a combination thereof. Those skilled in the art will readily appreciate that any macromolecule, including almost all proteins or peptides, can be used as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or may be expressed recombinantly. Antigens and/or antigenic determinants may be specific to a certain tissue, such as cancer cells, or they may be widely expressed. In addition, fragments of larger molecules may also serve as antigens. In one aspect, the antigen is a tumor antigen.

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 aspects, the antigen is displayed on the surface of the APC.

As used herein, "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor size, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an extension of the patient's overall or progression-free survival, an extension of the patient's expected life span, or an improvement in various tumor-related physiological symptoms in the patient. Anti-tumor effect can also refer to the prevention of tumor occurrence, such as a vaccine.

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

The term "autologous" refers to the individual from which any material, such as immune cells, is derived and the individual into which the material is later reintroduced. For example, autologous T-cell therapy involves administering to an individual T cells isolated from the same individual. 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 an individual T cells obtained from a donor other than the individual.

"Cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that will invade neighboring tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors. Examples of cancers that may 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 invention can be used to reduce the size of tumors arising from, for example, cancers including: bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or eye, 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 , adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem neuroglioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancers, or any combination thereof. A particular cancer may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancer refers to cancer that is not amenable to surgical intervention and that did not respond to chemotherapy or radiation therapy initially, or that has become unresponsive over time.

It should be understood that, where an aspect is described herein with the language “comprising”, other similar aspects described with the language “consisting of” and/or “consisting essentially of” are also provided.

As used herein, "cytokine" refers to a non-antibody protein released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with another cell to mediate a response in the other cell. Cytokines can be expressed endogenously by cells, added to cells in culture, administered to an individual, or any combination thereof. Cytokines can be released by immune cells including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic interleukins, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, while proinflammatory interleukins can promote inflammatory responses. Examples of homeostatic interleukins 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) γ. Examples of proinflammatory interleukins include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, 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).

"Cell chemokines" are a class of cytokines that mediate cell tropism or directional movement. Examples of cytokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived cytokine (MDC or CCL22), monocyte cytokine 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1a (MIP-1a, MIP-1a), MIP-1b (MIP-1b), gamma-inducing protein 10 (IP-10), and thymic and activation-regulated cytokine (TARC or CCL17).

Other examples of interleukins include, but are not limited to, interleukin (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific interleukin 3 (MCP3 or CCL7), monocyte cytokine 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), oncostatin M (OSM), CD 154, lymphotoxin (LT) β, 4-IBB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

The term "engineered autologous cell therapy" can be abbreviated as "eACT™", also known as adoptive cell transfer, is the process of collecting a patient's own immune cells, such as T cells and/or NK cells, and then genetically altering them to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant disease cells. Immune cells, such as 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, such as T cells or immune cells, are engineered to express an extracellular single chain variable fragment (scFv) specific for a specific tumor antigen, and the scFv is connected to an intracellular signaling portion comprising a co-stimulatory domain and an activation domain. The co-stimulatory domain can be derived from, for example, CD28, and the activation domain can be derived from, for example, CD3-ζ (Figure 1). In certain embodiments, CAR is designed to have two, three, four or more co-stimulatory domains. CAR scFv can be designed to target, for example, CD19, which is a transmembrane protein expressed by cells in the B cell lineage, which includes 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, and these references are incorporated by reference in their entirety.

As used herein, the term "Fas" refers to the Fas receptor protein or a portion thereof. 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 typical amino acid sequence of human Fas receptor is shown in Table 1 (UniProt P25445; SEQ ID NO: 30). The binding of Fas ligand to Fas receptor on cells leads to cell apoptosis. Some aspects of the present disclosure relate to immune cells modified to express one or more chimeric polypeptides, the one or more chimeric polypeptides comprising an extracellular ligand binding domain (Fas ECD) of a Fas receptor linked to a heterologous intracellular domain, such as a CD27 intracellular domain (e.g., SEQ ID NO: 22), a 4-1BB intracellular domain (e.g., SEQ ID NO: 23), or an OX40 intracellular domain (e.g., SEQ ID NO: 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 aspects, Fas is Fas dominant negative, or "FasDN". As used herein, "FasDN" refers to a Fas receptor modified to include a truncated Fas intracellular domain. Examples of FasDN sequences that can be used in the compositions and methods disclosed herein are presented in Table 1 (SEQ ID NO: 27). In some aspects, the chimeric Fas polypeptide further comprises a signal peptide. Any signal peptide that can promote the expression of the chimeric Fas polypeptide can be used in the compositions and methods disclosed herein. In some aspects, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 28. In some aspects, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 29. Table 1. Exemplary Fas constructs Fas-CD27 (Fas extracellular domain (ECD); CD27 intracellular domain (ICD) ) Fas-4-1BB (Fas ECD; 4-1BB ICD ) Fas-OX40-1 (Fas ECD; OX-40 ICD ) Fas-OX40-2 (Fas ECD; OX-40 ICD ) Fas-OX40-3 (Fas ECD; OX-40 ICD ) Fas-DN (Fas ECD; truncated Fas ICD ) Signal peptide 1 Signal peptide 2 Typical Fas receptor (UniProt P25445) (Signal peptide) Fas extracellular domain (ECD)

As understood in the art, "immune response" generally refers to the response of a vertebrate body to foreign or abnormal organisms, such as cancer cells, which protects the organism against these foreign organisms and the diseases caused by them. 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, interleukins, and complements) produced by any of these cells or the liver, which can selectively target, bind, damage, destroy, and/or eliminate from the vertebrate body invading pathogens, cells or tissues infected with pathogens, cancerous cells or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. An immune response includes, for example, activation or suppression of T cells, such as effector T cells, Th cells, CD4 ⁺ cells, CD8 ⁺ T cells, or Treg cells, or activation or suppression of any other cells of the immune system, such as NK cells. In some aspects, an immune response refers to NK cell-mediated killing of foreign cells, such as allogeneic T cell therapy.

"Immunotherapy" refers to the treatment of individuals who have a disease or are at risk of contracting a disease or experiencing a recurrence of a disease by methods that include inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

As used herein, the term "inactivating" or "inactivation" with respect to, for example, a gene or a protein refers to a measure that can induce a reduction in protein expression. In some aspects, inactivation can be achieved by the deletion or mutation of all or part of the coding region of a gene or all or part of the non-coding region of a gene, which results in a reduction in the expression of the gene or the protein encoded by the gene. In some aspects, inactivation is achieved by deleting the entire coding region of a gene. In some aspects, inactivation is achieved by a partial deletion of the coding region of a gene. In some aspects, inactivation is achieved by deleting one or more regulatory elements that promote gene expression. In some aspects, inactivation is achieved by a reduction in gene expression or loss of expression caused by a mutation of one or more regulatory elements. In some aspects, inactivation is achieved by a mutation of one or more nucleic acids that results in the expression of a non-functional protein. In some aspects, inactivation is achieved by expression of a non-functional protein due to a missense mutation. In some aspects, inactivation is achieved by reduced expression of a protein due to interference with the transcription or translation of a gene. In some aspects, reduced expression is relative to the expression of the target gene in the cell before modification (e.g., deletion or mutation). In some aspects, before modification, the expression of the gene is measured, then the cell is modified, and then the expression of the gene is measured after modification.

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

As used herein, the term "iPS cell" or "iPSC" refers to a cell that has been reverse-differentiated (or reprogrammed) to a more naive (e.g., pluripotent) state. A variety of methods are known to reverse-differentiate cells, including but not limited to overexpressing Oct3/4, Sox2, Klf4, and c-Myc ("Yamanaka factor") in cells (see, e.g., Takahashi and Yamanaka, Cell 126 : 663-76 (2006)). In some aspects, iPS cells are pluripotent cells, e.g., capable of differentiating into a limited number of cell types. In some aspects, iPS cells are omnipotent cells, e.g., capable of differentiating into any cell type. In some aspects, iPS cells can be redifferentiated into a specific type of cell, such as an immune cell.

As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (toxic to cells) lymphocytes that represent a major component of the innate immune system. NK cells reject tumors and virus-infected cells by inducing apoptosis or programmed cell death of target cells. NK cells are called "natural killers" because they do not require activation to kill target cells. T cells play a major role in cell-mediated immunity. The T cell receptor (TCR) expressed on the surface of T cells distinguishes T cells from other lymphocyte types. The thymus, as a special organ of the immune system, is primarily responsible for T cell maturation. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) memory TSC stem cells, such as naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (i) central memory TCM cells express L-selectin and CCR7, secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but instead produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells or CD4+CD25+ regulatory T cells), natural killer T cells (NKT); and γδ T cells.

B cells play a major role in humoral immunity (antibody involvement). B cells produce antibodies and antigens, and function as antigen presenting cells (APCs) and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence their name.

As used herein, "class I MHC molecule" refers to the protein product of a wild-type or variant class I HLA gene encoding a class I MHC molecule. Therefore, "class I HLA molecule" and "class I MHC molecule" can be used interchangeably herein. Class I MHC molecules contain two protein chains: the α chain and the 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 α chain of the class I MHC molecule is encoded by the HLA gene complex. The HLA complex is located in the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes with diverse functions. HLA genes are highly variable, with more than 20,000 HLA alleles and related alleles known to the art, including more than 15,000 class I HLA alleles, encoding thousands of HLA proteins, including more than 10,000 class I HLA proteins. There are at least three genes in the HLA complex that encode class I MHC alpha chain proteins: HLA-A, HLA-B, and HLA-C. In addition, HLA-E, HLA-F, and HLA-G encode proteins that bind to class I MHC molecules. Table 2. Amino acid sequence of human β2m SEQ ID NO: sequence twenty one MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

As used herein, "class II MHC molecules" refers to the protein products of wild-type or variant class II HLA genes encoding class II MHC molecules. Therefore, "class II MHC molecules" can be used interchangeably with "class II HLA molecules." Typical class II MHC molecules include two protein chains: an alpha chain and a beta chain. In general, the naturally occurring alpha chain and beta chain each include a transmembrane domain that anchors the alpha/beta chain to the cell surface; and an extracellular domain that carries antigen and interacts with the TCR and/or CD4 expressed on T cells. Both class II MHC alpha and beta chains are encoded by the HLA gene complex. The HLA complex is located in the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes with diverse functions. The HLA gene complex is highly variable, with more than 20,000 HLA alleles and related alleles known in the art, including more than 250 class II MHC α chain alleles and 5,000 class II MHC β chain alleles, encoding thousands of class II MHC proteins. Three loci in the HLA complex encode class II MHC proteins: HLA-DP, HLA-DQ, and HLA-DR. HLA-DO and HLA-DM encode proteins that bind to class II MHC molecules and support their configuration and function.

As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, dextrose), etc.), non-aqueous solvents (e.g. propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coating agents, 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, formulators, disintegrants, lubricants, sweeteners, flavor enhancers, dyes, fluids and nutritional supplements, as well as similar materials and combinations thereof known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

"Enhancing an endogenous immune response" means increasing the effectiveness or potency of an individual's existing immune response. This increase in effectiveness and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

As used herein, the term "recombinant" or "modified" cell is intended to refer to a cell that contains a nucleic acid that does not naturally occur in the cell, such as an immune cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to refer to a particular individual cell, but also to the progeny of such cells. Although certain modifications may occur in subsequent progeny due to mutations or environmental influences, such progeny are still included within the scope of the term "recombinant" or "modified" as used herein.

As used herein, the terms "reduced expression" and "increased expression" refer to the expression of a particular gene or protein in a cell relative to a control, for example, the expression of a particular gene in a modified cell relative to the expression of the gene in a wild-type (unmodified) cell. Relative expression can be based on mRNA levels and/or protein levels. Any method that measures mRNA and/or protein levels can be used to determine whether a gene or protein has decreased or increased expression, including but not limited to immunohistochemistry and PCR-based techniques.

In some aspects, a cell having "reduced expression" of a particular gene or protein has 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 level of the gene or protein in an unmodified cell, e.g., a wild-type cell of the same cell type. In some aspects, an unmodified cell, such as a wild-type cell of the same cell type, expresses a particular gene or protein, and the modified cell has no detectable level of protein or gene expression, such as a gene or protein "knocked out." Thus, the term "knockout" refers to the complete elimination of expression of a particular gene or protein, such that no detectable level of gene or protein expression is present in the cell.

In some aspects, a cell having "increased expression" or "overexpression" of a particular gene or protein has 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 210%, greater than about 220%, greater than about 230%, greater than about 240%, 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 420%, 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 More than about 180%, more than about 190%, more than about 200%, more than about 225%, more than about 250%, more than about 275%, more than about 300%, more than about 350%, more than about 400%, more than about 450%, more than about 500%, more than about 600%, more than about 700%, more than about 800%, more than about 900%, or more than about 1000% expression level, wherein 100% expression corresponds to the expression observed in the case of wild-type cells. In some aspects, a cell having "increased expression" or "overexpression" of a particular gene or protein has 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, compared to the expression of the gene or protein in an unmodified cell, e.g., a wild-type cell or cell population of the same cell type, or the same cell or cell population before modification. , 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 aspects, an unmodified cell, such as a wild-type cell of the same cell type or the same cell or cell population prior to modification, does not have expression of a particular gene or protein, and the increase in expression is any expression of the gene or protein.

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

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

As used herein, the terms "chimeric antigen receptor" and "CAR" refer to a recombinant fusion protein having an antigen-specific extracellular domain coupled to an intracellular domain, which directs cells to perform a specific function after the antigen binds to the extracellular domain. In some aspects, the chimeric antigen receptor disclosed herein comprises a chimeric polypeptide of the present disclosure.

As used herein, the term "T cell receptor" (TCR) refers to a heteromeric cell surface receptor capable of specifically interacting with a target antigen. As used herein, "TCR" includes, but is not limited to, naturally occurring and non-naturally occurring TCRs; full-length TCRs and antigen-binding portions thereof; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells and are responsible for T cell recognition and targeting of antigen presenting cells. Antigen presenting cells (APCs) display fragments of foreign proteins (antigens) complexed with major histocompatibility complexes (MHC; also referred to herein as complexed with HLA molecules, e.g., class 1 HLA molecules). TCRs recognize and bind antigen-HLA complexes and recruit CD3 (expressed by T cells), thereby activating the TCR. Activated TCR initiates downstream signaling and immune responses, including the destruction of antigen-presenting cells.

As used herein, "exogenous TCR" or "eTCR" refers to a TCR that is heterologous to the cell expressing the TCR. As used herein, the term "heterologous" refers to a substance that is not natural or naturally present, such as in a particular cell.

In general, TCR may comprise two chains, i.e., (i) one α chain and one β chain (α-β TCR) for α-β T cells, or (ii) one γ chain and one δ chain (γ-δ TCR) for γ-δ T cells, which are interconnected by a disulfide bond. Each chain comprises a variable domain (α chain variable domain, β chain variable domain, γ chain variable domain, and δ chain variable domain) and a constant region (α chain constant region, β chain constant region, γ chain constant region, and δ chain constant region). The variable domain is located at the distal end of the cell membrane, and the variable domain interacts with the antigen. The constant region is located at the proximal end of the cell membrane. TCR may further comprise a transmembrane region and a short cytoplasmic tail region. As used herein, the term "constant region" encompasses the transmembrane region and the cytoplasmic tail (when present), as well as the traditional "constant region".

The variable domains can be further subdivided into hypervariable regions, called complement determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each alpha chain variable domain 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 on each chain are involved in antigen binding, CDR3 is believed to be the major antigen binding region. CDR1 also interacts with antigen, while CD2 is believed to primarily recognize the ELLA complex. In one embodiment, the γ chain variable domain includes Vγ1, Vγ2, Vγ3, Vγ4, Vγ5, Vγ6, Vγ7, Vγ8 and Vγ9, and examples of the δ chain variable domain include Vδ1, Vδ2, Vδ3, Vδ4, Vδ5, Vδ6, Vδ7, Vδ8 and Vδ9. The combination of specific γ chain variable domains and δ chain variable domains in the TCR is not limited, for example, the eTCR is any one of Vγ3-Vδ1 TCR (g3d1TCR), Vγ4-Vδ1 TCR (g4d1TCR), Vγ9-Vδ1 TCR (g9d1TCR) and Vγ9 -Vδ2 TCR (g9d2TCR).

If not expressly specified, and unless the context indicates otherwise, the term "TCR" also includes antigen-binding fragments or antigen-binding portions of any TCR disclosed herein, and includes 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" further refers to TCRs described herein that are expressed on the surface of cells other than T cells (e.g., cells that naturally express or are modified to express CD3 as described herein), or TCRs described herein that do not contain a cell membrane (e.g., an isolated TCR or a soluble TCR).

"TCR fragment", "antigen binding molecule" or "portion of a TCR" refers to any portion of a TCR that is smaller than the entire TCR. The antigen binding molecule may include an antigenic complementary determining region (CDR).

"Suicide gene" refers to a gene that causes a cell to commit suicide. In some embodiments, the suicide gene causes the cell to commit suicide by apoptosis. Non-limiting examples of suicide genes include viral thymidine kinase, cytosine deaminase, intracellular antibodies against antioxidant enzymes (AOE), bacterial nitroreductase, caspase, and DNase.

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 further phosphorylated to deoxythymidine diphosphate and then to deoxythymidine triphosphate by viral thymidine kinase and nucleoside diphosphate kinase, 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), can terminate DNA synthesis after incorporation into synthesized DNA. The termination of synthesis triggers the cell apoptosis signaling cascade. Although 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 synthesis arrest and cell apoptosis. Although variants of HSV-TK are not restricted, HSV-TK is, for example, TK007 (see Preuss et al., Hum Gene Ther. 2010 Aug; 21(8): 929-41).

In some embodiments, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyzes cytosine to uracil and releases ammonia. Under physiological conditions, the modified site is recognized by the nuclease endonuclease, followed by the cleavage of the phosphodiester bond in the DNA, and repair is initiated by the incorporation of 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 to the more toxic 5-FU (thymidylate synthase suicide inhibitor), thereby inhibiting cell growth and cell apoptosis.

As used herein, the term "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or enhances the health of an individual in the medical treatment of a disease. This includes, but is not limited to, a reduction in the frequency or severity of disease signs or symptoms.

As used herein, the term "treating" or "treatment" of a disease or disorder refers to the implementation of a regimen that may include administering one or more therapies to a patient in an effort to reduce the signs or symptoms of the disease. In some aspects, treatment reduces the rate of disease progression, improves or alleviates the disease state, and/or promotes relief or improved prognosis. Reduction can occur before signs or symptoms of the disease or disorder appear as well as after they occur. Thus, in some aspects, "treatment" includes "preventing" or "prevention" of a disease or undesirable condition. However, "treatment" does not require complete relief of all signs and/or symptoms, does not require a cure, and specifically includes regimens that have only a marginal effect on the patient.

In various aspects, the modified cell populations described herein can be used to treat a disease in an individual in need or to alleviate symptoms associated with a disease (e.g., cancer). In some aspects, the modified cells are immune cells or iPS cells transduced with CAR or TCR and are further modified to exhibit reduced expression of endogenous class I MHC and class II MHC HLA genes in the cells and increased expression or expression of certain heterologous genes, thereby making such modified cells less susceptible to being killed by the individual's immune system when such cells are administered to the individual.

As used herein, the terms "ug" and "uM" may be used interchangeably with "μg" and "μΜ", respectively.

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 relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

Units, suffixes and symbols are expressed in the form recognized by the International System of Units (Système International de Unites, SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any concentration range, percentage range, ratio range or integer range described herein should be understood to include any integer value within the range and, where appropriate, its fractions (such as one tenth and one hundredth of an integer).

The abbreviations used herein are defined throughout this disclosure. Various aspects of this disclosure will be described in further detail in the following subsections.

Each aspect described herein will be described in further detail in the following subsections. II. Compositions of the Disclosure

Some aspects of the present disclosure relate to modified 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) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (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.

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

Other aspects of the present disclosure relate to a modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Other aspects of the present disclosure relate to a modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding MHC-II human leukocyte antigen (HLA); and (iii) an inactivated endogenous gene encoding CD58.

Other aspects of the present disclosure relate to a modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Other aspects of the present disclosure relate to a modified cell comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Other aspects of the disclosure relate to dominant negative Fas (Fas-DN), Fas-CD27 chimeric polypeptide (Fas-CD27), Fas-4-1BB chimeric polypeptide (Fas-BB), or Fas-OX40 chimeric polypeptide (Fas-OX40).

Other aspects of the present disclosure relate to a guide RNA capable of hybridizing with the human CD58 gene. II.A. Modified Cells

Some aspects of the present disclosure relate to modified 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) relative to wild-type cells of the same cell type; (iii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (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.

In some aspects, the cells disclosed herein have increased persistence in vivo relative to wild-type cells of the same cell type. In some aspects, the cells disclosed herein are less immunogenic when administered to a human subject. In some aspects, after administration of the cells, the human subject has less immune response to the cells. In some aspects, after administration of the cells, the human subject has reduced killing of the cells by NK cells. In some aspects, NK cell-mediated killing of the cells 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% following administration of the cells relative to wild-type cells of the same cell type administered to a human subject.

In some aspects, the MHC-I human leukocyte antigens are HLA-A, HLA-B, and HLA-C. In some aspects, the MHC-I human leukocyte antigens are expressed in an amount 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 amount of MHC-I human leukocyte antigen expression in a wild-type cell of the same cell type. In some aspects, the cells have no detectable MHC-I human leukocyte antigen expression.

In some aspects, the reduced expression of endogenous MHC-I human leukocyte antigens is caused by a mutation or deletion of one or more endogenous genes encoding beta-2-microglobulin (B2M). In some aspects, the 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 aspects, the cells have no detectable B2M expression.

In some aspects, the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ, and HLA-DR. In some aspects, the MHC-II human leukocyte antigens are expressed at less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the amount of MHC-II human leukocyte antigen expressed in wild-type cells of the same cell type. In some aspects, the cells do not have detectable expression of MHC-II human leukocyte antigens.

In some aspects, the reduced expression of endogenous MHC-II human leukocyte antigens is caused by a mutation or deletion of one or more endogenous genes encoding class II major histocompatibility complex transactivator protein (CIITA). In some aspects, 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 aspects, the cells have no detectable CIITA expression.

In some aspects, the cell further comprises reduced endogenous CD58 expression relative to wild-type cells of the same cell type. In some aspects, reduced expression of CD58 is caused by a mutation or deletion of one or more endogenous genes encoding CD58. In some aspects, the amount of CD58 expressed 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 amount of CD58 expressed in wild-type cells of the same cell type. In some aspects, the cells have no detectable functional CD58 expression. II.A.1. Expression of Fas Constructs

In some aspects, the cell further comprises a nucleic acid encoding a Fas-DN (e.g., a Fas-DN disclosed herein). Thus, some aspects of the disclosure relate to a modified cell, such as an immune cell, comprising: (i) a CAR or an eTCR; (ii) an endogenous MHC-I human leukocyte antigen whose expression is reduced relative to a wild-type cell of the same cell type; (iii) an endogenous MHC-II human leukocyte antigen whose expression is reduced relative to a wild-type cell of the same cell type; and (iv) Fas-DN. In some aspects, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) an endogenous B2M whose expression is reduced; (iii) an endogenous CIITA whose expression is reduced; and (iv) Fas-DN. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) reduced expression of endogenous B2M; (iii) reduced expression of endogenous CIITA; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; and (v) Fas-DN.

In some aspects, 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 shown in SEQ ID NO: 35, 36, or 40. In some aspects, Fas-DN is encoded by the nucleotide sequence shown in SEQ ID NO: 35, 36, or 40. Table 3: Encoding sequence SEQ ID NO describe sequence 33 CAG-HLA-E GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCC CTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTC CGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGC GTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAG GGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTC TCATCATTTTGGaCAAAGAATTCCatcgatGCCACCATGTCCCGCTCCGTGGCACTGGCGGTATTGGCCCTGTTGTCACTGAGCGGCCTGGAGGCAGTCATGGCTCCTCGGACCCTGATCCTGGGTGGAGGGGGCTCCGGAGGGGGAGGTTCTGGGGGTGGAGGCAGTATTCAGCGGACCCCTAAAATTCAAGTTTATTCAAGGCACCCTGCAGAAAATGGCAAAAGTAATTTCCTTAATTGCTACGTGTCCGGTTTCCACCCCTCCGATATAGAAGTAGATCTTCTGAAGAACGGAGAACGGATTGAGAAGGTGGAACACAGTGACCTGAGCTTCAGTAAGGATTGGTCCTTCTACCTCCTGTATTATACTGAATTTACACCAACTGAAAAGGACGAGTACGCCTGCAGGGTCAACCATGTGACTT TGTCCCAGCCAAAGATCGTGAAATGGGATCGCGACATGGGAGGGGGGTCAGGGGGCGGAGGGTCAGGCGGCGGTGGGAGTGGTGGGGGAGGAAGCGGCTCTCATTCCCTTAAATATTTCCATACAAGCGTGTCTCGACCCGGCAGGGGTGAACCTCGCTTCATCTCCGTTGGTTATGTCGATGATACCCAGTTTGTCCGGTTTGATAACGACGCAGCAAGCCCCAGGATGGTGCCCAGAGCTCCGTGGATGGAGCAGGAGGGGTCTGAGTACTGGGATAGAGAAACAAGAAGTGCCAGGGACACTGCACAGATCTTTCGAGTGAATCTGAGGACCCTCAGGGGCTACTATAACCAATCTGAGGCGGGAAGCCACACCCTGCAGTGGATGCATGGATGCGAGCTGGGGCCCGATAGACGGTTTTTG CGGGGATATGAGCAGTTTGCGTATGACGGCAAGGATTATCTGACGCTCAATGAGGACCTCAGAAGTTGGACGGCCGTTGATACCGCCGCCCAGATCAGCGAACAGAAAAGCAACGATGCGTCCGAAGCCGAGCACCAGCGGGCTTACCTGGAGGACACCTGTGTCGAGTGGCTGCATAAATACCTCGAGAAGGGAAAGGAAACACTGCTGCACCTGGAGCCTCCTAAGACTCACGTAACTCATCATCCTATTAGCGATCACGAGGCCACTTTGCGGTGTTGGGCTCTGGGATTCTACCCAGCTGAAATTACATTGACATGGCAACAGGATGGAGAGGGGCATACCCAGGACACGGAGCTCGTGGAAACACGCCCTGCCGGTGATGGCACCTTTCAGAAGTGGGCAGCGGTGGTGGTGCCTAGCGGCG AGGAGCAGCGATATACTTGCCATGTGCAGCATGAAGGACTTCCCGAACCCGTTACACTGCGCTGGAAACCGGCTTCCCAGCCGACAATACCCATCGTGGGTATCATCGCCGGGCTCGTGCTCCTCGGCAGCGTTGTGTCAGGCGCAGTGGTGGCAGCCGTGATCTGGCGAAAGAAATCCAGTGGCGGCAAAGGCGGGAGCTACTATAAAGCTGAATGGTCCGACAGCGCCCAGGGGAGCGAGAGTCACTCTTTGTGAGAATTCGAGCTCATCGATGCATGGTACCCGGGCATGCTCGAGCTAGCAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctca 34 CAG-tEGFR-2A-HSVTK GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTC CGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG GGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG GGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGG GCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCGGC CCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGG ACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGaCAAAGAATTCCatcgatGCCACCATGGCTAGCTATCCATGTCACCAGCACGCAAGCGCGTTTGACCAGGCGGCACGAAGCCGAGGCCACAGCAACCGCAGAACAGC GCTTCGGCCTCGCCGACAGCAAGAGGCCACAGAGGTTAGATTGGAACAGAAAATGCCAACATTGCTCCGGGTCTACATTGACGGGCCCCATGGCATGGGAAAAACCACTACAACTCAACTCCTCGTGGCACTCGGGTCACGCGATGACATAGTTTATGTCCCCGAGCCAATGACGTATTGGCAGGTGCTGGGGGCCTCAGAGACTATTGCCAACATCTACACAACGCAACATAGATTGGACCAAGGGGAGATTAGCGCAGGA GACGCTGCAGTTGTCATGACTTCCGCTCAAATCACCATGGGAATGCCCTACGCAGTCACAGACGCTGTTCTGGCGCCCCACATCGGGGGCGAAGCTGGATCCAGTCACGCGCCACCACCTGCACTTACTTTGATCTTTGATCGACATCCTATAGCGCATCTCCTCTGTTATCCTGCAGCCCGCTACTTGATGGGATCAATGACTCCACAAGCCGTTCTGGCCTTTGTGGCGCTTATACCGCCTACACTCCCCGGCACGAAT ATAGTACTTGGAGCTCTTCCTGAAGACAGGCACATTGATAGACTGGCTAAAAGGCAGCGCCCAGGGGAGAGGCTTGATTTGGCGATGCTTGCAGCCATTCGACGAGTATATGGGTTGCTTGCAAACACTGTACGGTATCTGCAAGGAGGCGGAAGTTGGAGAGAGGACTGGGGGCAGTTGAGCGGGACAGCCGTTCCCCCCCAAGGCGCTGAACCTCAATCTAACGCTGGTCCACGCCCTCATATAGGGGACACATTGTTC ACCTTGTTCAGGGCCCCCGAGTTGCTGGCCCCTAACGGCGATTTGTATAATGTTTTCGCTTGGGCCCTTGACGTACTTGCCAAGAGGCTGAGACCAATGCATGTATTTATACTGGACTACGATCAAAGCCCTGCTGGATGTCGCGACGCCTTGTTGCAGCTGACCAGCGGGATGGTCCAGACGCATGTCACGACGCCGGGTTCCATACCAACGATATGCGATCTTGCTCGAACTTTTGCTAGGGAGATGGGCGAGGCTAAC GGTAGTGGTGAGGGCAGAGGGTCACTGCTGACGTGTGGGGATGTAGAGGAAAATCCGGGTCCGatgctcctgctggttacctctctgctcctgtgtgaactgcctcatcctgcattcctgctcattccccggaaagtttgtaacggcatcgggattggtgaatttaaagactctctgtctatcaacgccactaatatcaaacactttaaaaactgtacttccatctccggagatctgcatatactgcccgttgcttttag gagactcctttacccacacaccaccactcgacccccaagaactggatatcctgaaaactgttaaggagattacaggcttcctgctgattcaggcctggcctgagaaccgcactgatctgcatgctttcgagaacctggaaattattaggggaaggaccaaacaacacggacagttcagcctcgccgttgtgtctctcaacattacatctctcggactccgctccctcaaagaaatctccgacggcgacgtcattatcagcg gcaacaaaaatctctgttatgctaatactatcaactggaagaagctgtttggaaccagcggacaaaaaacaaaaataatttctaaccggggggaaaattcttgcaaagctactggacaagtgtgccacgccctgtgtagccccgagggatgctggggacccgaacctcgcgactgtgttagctgtagaaatgtgtcccgcggaagagaatgtgttgacaaatgtaacctgctggaaggagaacctagggaatttgtggaaaa ctctgagtgcattcaatgtcaccccgagtgtctgcctcaagccatgaacataacttgcacaggccgcggacccgacaattgtatccagtgtgctcattacattgacggacctcattgcgtgaaaacttgtccagccggcgtcatgggagagaataacacactcgtttggaagtatgctgacgcaggtcatgtctgtcacctctgtcaccctaactgcacatacggttgcactggtcccggtctcgaaggatgtcctactaa tggccctaaaattccaagcattgctacaggcatggttggcgccctgctcctgctcctcgttgtcgctctcgggattggtctctttatgTAAGAATTCGAGCTCATCGATGCATGGTACCCGGGCATGCTCGAGCTAGCAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctca 35 CAG-FasDN GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTT TCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGCGGCGGGCGGGAGTCGCT GCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGC GAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCG GCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTT TCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGaCAAAGAATTCCatcgatGCCACCatgctcggcatctggacgctgctgccccttgtacttacctccgtggctcggctttctagtaaatcagtgaatgctcaggtcacagacatcaactccaaaggcttggaactgaggaaaaccgtgaccaccgtggagacacagaacctggaggggctgcaccacgatggccagttctgccataaaccttgtccccccggcgaaagaaaagcgagggactgtaccgtgaacggcgatgagccagattgcgtgccttgccag gaaggtaaggagtacaccgataaagctcatttttcctcaaagtgtcggaggtgtaggttgtgcgatgaaggacatgggctcgaagtggaaattaactgcacccgcacacagaacactaagtgtaggtgcaaaccgaatttcttctgcaactccacggtgtgcgaacactgtgacccctgtacaaagtgcgagcatggcattattaaggagtgtacccttacatcaaatacaaagtgtaaggaagaaggttctaggagcaacttgggctggctgtgcctcctgctgctgcctataccccttattgtttgggttaagcggaaggaagtgcagaaaa cttgtagaaaacaccgcaaggagaaccagggcagtcatgagtccccaacgctcaatcctgagactgtggccataaacctgagcgatgtcgatctcattctcaaggatatcacaagcgacagcgagaatagtaatttccggaatgagatccagagtctggtgtgaGAATTCGAGCTCATCGATGCATGGTACCCGGGCATGCTCGAGCTAGCAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtctctca 36 HLA-E-HSVTK-FasDN ATGTCCCGCTCCGTGGCACTGGCGGTATTGGCCCTGTTGTCACTGAGCGGCCTGGAGGCAGTCATGGCTCCTCGGACCCTGATCCTGGGTGGAGGGGGCTCCGGAGGGGGAGGTTCTGGGGGTGGAGGCAGTATTCAGCGGACCCCTAAAATTCAAGTTTATTCAAGGCACCCTGCAGAAAATGGCAAAAGTAATTTCCTTAATTGCTACGTGTCCGGTTTCCACCCCTCCGATATAGAAGTAGATCTTCTGAAGAACGGAGAACGGATTGAGAAGGTGGAACACAGTGACCTGAGCTTCAGTAAGGATTGGTCCTTCTACCTCCTGTATTATACTGAATTTACACCAACTGAAAAGGACGAGTACGCCTGCAGGGTCAACCATGTGACTTTGTCCCAGCCAAAGATCGTGAAATGGGATCGCGACATG GGAGGAGGGGGGTCAGGGGGCGGAGGGTCAGGCGGCGGTGGGAGTGGTGGGGGAGGAAGCGGCTCTCATTCCCTTAAATATTTCCATACAAGCGTGTCTCGACCCGGCAGGGGTGAACCTCGCTTCATCTCCGTTGGTTATGTCGATGATACCCAGTTTGTCCGGTTTGATAACGACGCAGCAAGCCCCAGGATGGTGCCCAGAGCTCCGTGGATGGAGCAGGAGGGGTCTGAGTACTGGGATAGAGAAACAAGAAGTGCCAGGGACACTGCACAGATCTTTCGAGTGAATCTGAGGACCCTCAGGGGCTACTATAACCAATCTGAGGCGGGAAGCCACACCCTGCAGTGGATGCATGGATGCGAGCTGGGGCCCGATAGACGGTTTTTGCGGGGATATGAGCAGTTTGCGTATGACGGCAAGGATTAT CTGACGCTCAATGAGGACCTCAGAAGTTGGACGGCCGTTGATACCGCCGCCCAGATCAGCGAACAGAAAAGCAACGATGCGTCCGAAGCCGAGCACCAGCGGGCTTACCTGGAGGACACCTGTGTCGAGTGGCTGCATAAATACCTCGAGAAGGGAAAGGAAACACTGCTGCACCTGGAGCCTCCTAAGACTCACGTAACTCATCATCCTATTAGCGATCACGAGGCCACTTTGCGGTGTTGGGCTCTGGGATTCTACCCAGCTGAAATTACATTGACATGGCAACAGGATGGAGAGGGGCATACCCAGGACACGGAGCTCGTGGAAACACGCCCTGCCGGTGATGGCACCTTTCAGAAGTGGGCAGCGGTGGTGGTGCCTAGCGGCGAGGAGCAGCGATATACTTGCCATGTGCAGCATGAAGGACTT CCCGAACCCGTTACACTGCGCTGGAAACCGGCTTCCCAGCCGACAATACCCATCGTGGGTATCATCGCCGGGCTCGTGCTCCTCGGCAGCGTTGTGTCAGGCGCAGTGGTGGCAGCCGTGATCTGGCGAAAGAAATCCAGTGGCGGCAAAGGCGGGAGCTACTATAAAGCTGAATGGTCCGACAGCGCCCAGGGGAGCGAGAGTCACTCTTTGGGTTCTGGTGCTACAAATTTTTCACTTCTTAAACAGGCTGGCGACGTAGAAGAAAACCCCGGTCCTATGCTCGGTATCTGGACCCTGCTGCCTCTCGTCCTCACCTCTGTTGCCCGCCTTTCTAGCAAGTCCGTAAACGCCCAGGTCACAGACATCAATTCAAAAGGCCTTGAACTTCGAAAAACCGTCACCACCGTAGAAACACAGAATCTGGAA GGCCTGCACCACGATGGTCAATTTTGCCACAAGCCCTGTCCCCCCGGTGAACGCAAAGCCCGAGATTGCACAGTGAACGGTGATGAACCTGACTGCGTACCTTGTCAAGAGGGAAAAGAATACACCGATAAAGCCCATTTCAGTTCCAAATGTAGAAGGTGCCGACTCTGCGATGAAGGTCACGGGTTGGAAGTCGAAATCAACTGTACAAGAACACAAAATACCAAATGTCGCTGTAAACCTAATTTCTTCTGTAACTCAACTGTATGTGAACACTGCGATCCATGCACAAAATGCGAGCATGGCATAATAAAGGAATGCACTCTGACATCTAACACAAAATGTAAAGAAGAAGGGTCTCGCTCCAACCTTGGATGGCTTTGCCTCCTGCTGCTCCCTATCCCCCTTATCGTGTGGGTTAAACGCAAA GAAGTTCAAAAAACATGTCGAAAACACAGAAAAGAAAATCAAGGAAGCCACGAATCACCAACACTTAATCCCGAAACTGTAGCCATTAATCTTAGCGATGTTGACCTTATACTCAAAGACATAACCTCTGATAGCGAAAACTCTAACTTCCGAAACGAAATCCAATCACTTGTTGGCTCAGGCGCAACAAACTTCTCTCTCTTGAAACAAGCAGGCGATGTCGAAGAGAACCCAGGTCCTATGGCAAGCTACCCCGGCCATCAGCATGCCTCTGCATTCGATCAGGCCGCTCGGTCAAGAGGTCATAGCAACCGCAGAACCGCGCTCAGACCCCGCAGGCAGCAGGAGGCTACCGAGGTGAGACCTGAACAGAAAATGCCCACACTGCTGAGAGTCTATATTGATGGTCCACATGGAATGGGAAAAACC ACGATCTACGTAACCGATGCTGTAGCCCTTGGGAGTAGAGATGACATCGTTTATGTACCTGAACCTATGACTTATTGGAGAGTGCTTGGAGCCAGCGAGACCATTGCAAACATTTACACTACTCAGCACCGCCTGGACCAGGGTGAGATTAGCGCAGGCGATGCCGCAGTGGTCATGACTTCAGCCCAAATCACGATGGGGATGCCATACGCCGTAACCGATGCTGTTCTGGCCCCACATATCGGCGGAGGCCGGCTCATCCCATGCCCCCCCACCAGCTTTGACTCTGATCTTTGACAGACACCCTATTGCAGCCTTGCTCTGTTATCCTGCTGCCCGGTACCTGATGGGCTCAATGACTCCTCAAGCTGTACTGGCTTTCGTTGCTCTCATTCCGCCCACCCTGCCTGGTACTAACATCGTTCTGGGA GCTCTGCCCGAGGATAGGCACATAGATAGACTCGCCAAACGCCAAAGGCCAGGAGAACGCTTGGACCTCGCCATGCTGGCCGCAATTAGACGAGTTTACGGGCTGCTCGCAAATACCGTGAGATACCTTCAATGCGGAGGAAGTTGGAGAGAAGATTGGGGGCAACTGTCTGGAACTGCTGTACCTCCACAGGGAGCTGAGCCACAATCTAATGCAGGACCGAGACCACACATTGGAGATACCTTGTTCACCTTGTTCAGAGCTCCCGAACTGCTGGCACCAAATGGAGATCTGTACAATGTTTTCGCGTGGGCCCTCGACGTGCTGGCGAAACGCCTGAGGAGTATGCACGTATTTATCTTGGATTATGACCAATCACCTGCTGGATGCCGAGACGCCCTGCTCCAACTTACTTCTGGTATGGTTCAA 37 CAG promoter GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCCCC ACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCC TTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTG CGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG CGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGaCAAAGAATT 38 HLA-E fusion ATGTCCCGCTCCGTGGCACTGGCGGTATTGGCCCTGTTGTCACTGAGCGGCCTGGAGGCAGTCATGGCTCCTCGGACCCTGATCCTGGGTGGAGGGGGCTCCGGAGGGGGAGGTTCTGGGGGTGGAGGCAGTATTCAGCGGACCCCTAAAATTCAAGTTTATTCAAGGCACCCTGCAGAAAATGGCAAAAGTAATTTCCTTAATTGCTACGTGTCCGGTTTCCACCCCTCCGATATAGAAGTAGATCTTCTGAAGAACGGAGAACGGATTGAGAAGGTGGAACACAGTGACCTGAGCTTCAGTAAGGATTGGTCCTTCTACCTCCTGTATTATACTGAATTTACACCAACTGAAAAGGACGAGTACGCCTGCAGG GTCAACCATGTGACTTTGTCCCAGCCAAAGATCGTGAAATGGGATCGCGACATGGGAGGGGGGTCAGGGGGCGGAGGGTCAGGCGGCGGTGGGAGTGGTGGGGGAGGAAGCGGCTCTCATTCCCTTAAATATTTCCATACAAGCGTGTCTCGACCCGGCAGGGGTGAACCTCGCTTCATCTCCGTTGGTTATGTCGATGATACCCAGTTTGTCCGGTTTGATAACGACGCAGCAAGCCCCAGGATGGTGCCCAGAGCTCCGTGGATGGAGCAGGAGGGGTCTGAGTACTGGGATAGAGAAACAAGAAGTGCCAGGGACACTGCACAGATCTTTCGAGTGAATCTGAGGACCCTCAGGGGCTACTATAACCAAT CTGAGGCGGGAAGCCACACCCTGCAGTGGATGCATGGATGCGAGCTGGGGCCCGATAGACGGTTTTTGCGGGGATATGAGCAGTTTGCGTATGACGGCAAGGATTATCTGACGCTCAATGAGGACCTCAGAAGTTGGACGGCCGTTGATACCGCCGCCCAGATCAGCGAACAGAAAAGCAACGATGCGTCCGAAGCCGAGCACCAGCGGGCTTACCTGGAGGACACCTGTGTCGAGTGGCTGCATAAATACCTCGAGAAGGGAAAGGAAACACTGCTGCACCTGGAGCCTCCTAAGACTCACGTAACTCATCATCCTATTAGCGATCACGAGGCCACTTTGCGGTGTTGGGCTCTGGGATTCTACCCAGCTGAAAT TACATTGACATGGCAACAGGATGGAGAGGGGCATACCCAGGACACGGAGCTCGTGGAAACACGCCCTGCCGGTGATGGCACCTTTCAGAAGTGGGCAGCGGTGGTGGTGCCTAGCGGCGAGGAGCAGCGATATACTTGCCATGTGCAGCATGAAGGACTTCCCGAACCCGTTACACTGCGCTGGAAACCGGCTTCCCAGCCGACAATACCCATCGTGGGTATCATCGCCGGGCTCGTGCTCCTCGGCAGCGTTGTGTCAGGCGCAGTGGTGGCAGCCGTGATCTGGCGAAAGAAATCCAGTGGCGGCAAAGGCGGGAGCTACTATAAAGCTGAATGGTCCGACAGCGCCCAGGGGAGCGAGAGTCACTCTTTGTAA 39 LNGFR-2A-HSVTK ATGGGAGCCGGAGCTACCGGCAGGGCAATGGACGGCCCAAGGCTTCTCTTGCTGCTGTTGCTTGGAGTCTCTCTGGGCGGGGCAAAGGAGGCGTGTCCTACTGGCCTTTATACGCACAGCGGGGAATGCTGTAAAGCGTGCAACCTCGGCGAAGGCGTAGCCCAACCATGCGGGGCCAACCAGACTGTGTGCGAGCCATGTCTTGACAGCGTCACGTTCAGCGACGTGGTGTCCGCGACCGAACCCTGCAAGC CATGTACGGAGTGCGTCGGGCTCCAAAGTATGAGCGCACCATGTGTGGAGGCTGACGACGCGGTATGCCGCTGTGCTTACGGTTATTACCAGGACGAGACGACGGGGCGCTGCGAAGCATGTCGAGTGTGCGAGGCCGGGTCTGGGCTTGTTTTCAGTTGCCAGGATAAGCAGAACACTGTATGTGAAGAATGCCCTGATGGTACGTACTCTGACGAAGCAAATCATGTAGATCCTTGTCTTCCGTGCACGGTC TGCGAAGACACAGAAAGACAGCTTAGAGAATGTACACGGTGGGCCGACGCGGAATGTGAGGAAATACCTGGGCGCTGGATTACGCGGAGTACCCCACCAGAGGGCTCAGATTCTACGGCCCCGAGCACACAAGAACCTGAGGCCCCCCCAGAACAGGATTTGATAGCTTCCACTGTTGCGGGGGTCGTCACCACTGTAATGGGCTCATCTCAACCTGTTGTCACACGGGGAACAACTGACAATCTCATACCAGT ATATTGCAGCATTCTTGCTGCCGTAGTGGTTGGGTTGGTCGCCTATATTGCCTTCAAAAGACGCGCGAAGAGGTCCGGTAGTGGTGAGGGCAGAGGGTCACTGCTGACGTGTGGGGATGTAGAGGAAAATCCGGGTCCGATGGCTAGCTATCCATGTCACCAGCACGCAAGCGCGTTTGACCAGGCGGCACGAAGCCGAGGCCACAGCAACCGCAGAACAGCGCTTCGGCCTCGCCGACAGCAAGAGGCCACAG AGGTTAGATTGGAACAGAAAATGCCAACATTGCTCCGGGTCTACATTGACGGGCCCCATGGCATGGGAAAAACCACTACAACTCAACTCCTCGTGGCACTCGGGTCACGCGATGACATAGTTTATGTCCCCGAGCCAATGACGTATTGGCAGGTGCTGGGGGCCTCAGAGACTATTGCCAACATCTACACAACGCAACATAGATTGGACCAAGGGGAGATTAGCGCAGGAGACGCTGCAGTTGTCATGACTTCC GCTCAAATCACCATGGGAATGCCCTACGCAGTCACAGACGCTGTTCTGGCGCCCCACATCGGGGGCGAAGCTGGATCCAGTCACGCGCCACCACCTGCACTTACTTTGATCTTTGATCGACATCCTATAGCGCATCTCCTCTGTTATCCTGCAGCCCGCTACTTGATGGGATCAATGACTCCACAAGCCGTTCTGGCCTTTGTGGCGCTTATACCGCCTACACTCCCCGGCACGAATATAGTACTTGGAGCTCT TCCTGAAGACAGGCACATTGATAGACTGGCTAAAAGGCAGCGCCCAGGGGAGAGGCTTGATTTGGCGATGCTTGCAGCCATTCGACGAGTATATGGGTTGCTTGCAAACACTGTACGGTATCTGCAAGGAGGCGGAAGTTGGAGAGAGGACTGGGGGCAGTTGAGCGGGACAGCCGTTCCCCCCCAAGGCGCTGAACCTCAATCTAACGCTGGTCCACGCCCTCATATAGGGGACACATTGTTCACCTTGTTCA GGGCCCCCGAGTTGCTGGCCCCTAACGGCGATTTGTATAATGTTTTCGCTTGGGCCCTTGACGTACTTGCCAAGAGGCTGAGACCAATGCATGTATTTATACTGGACTACGATCAAAGCCCTGCTGGATGTCGCGACGCCTTGTTGCAGCTGACCAGCGGGATGGTCCAGACGCATGTCACGACGCCGGGTTCCATACCAACGATATGCGATCTTGCTCGAACTTTTGCTAGGGAGATGGGCGAGGCTAACTAA 40 Fas-DN ATGCTCGGCATCTGGACGCTGCTGCCCCTTGTACTTACCTCCGTGGCTCGGCTTTCTAGTAAATCAGTGAATGCTCAGGTCACAGACATCAACTCCAAAGGCTTGGAACTGAGGAAAACCGTGACCACCGTGGAGACACAGAACCTGGAGGGGCTGCACCACGATGGCCAGTTCTGCCATAAACCTTGTCCCCCCGGCGAAAGAAAAGCGAGGGACTGTACCGTGAACGGCGATGAGCCAGATTGCGTGCCTTGCCAGGAAGGTAAGGAGTACACCGATAAAGCTCATTTTTCCTCAAAGTGTCGGAGGTGTAGGTTGTGCGATGAAGGACATGGGCTCGAAGTGGAAATTAACTGCACCCGCACACAGAACACTAAG TGTAGGTGCAAACCGAATTTCTTCTGCAACTCCACGGTGTGCGAACACTGTGACCCCTGTACAAAGTGCGAGCATGGCATTATTAAGGAGTGTACCCTTACATCAAATACAAAGTGTAAGGAAGAAGGTTCTAGGAGCAACTTGGGCTGGCTGTGCCTCCTGCTGCTGCCTATACCCCTTATTGTTTGGGTTAAGCGGAAGGAAGTGCAGAAAACTTGTAGAAAACACCGCAAGGAGAACCAGGGCAGTCATGAGTCCCCAACGCTCAATCCTGAGACTGTGGCCATAAACCTGAGCGATGTCGATCTCATTCTCAAGGATATCACAAGCGACAGCGAGAATAGTAATTTCCGGAATGAGATCCAGAGTCTGGTGTGA

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-DN. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-DN. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous CD58 whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-DN.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-DN. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-DN. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-DN.

In some aspects, the cell further comprises Fas-CD27, such as Fas-CD27 disclosed herein. Thus, some aspects of the disclosure relate to a modified cell, such as an immune cell, comprising: (i) a CAR or an eTCR; (ii) reduced expression of an endogenous MHC-I human leukocyte antigen relative to a wild-type cell of the same cell type; (iii) reduced expression of an endogenous MHC-II human leukocyte antigen relative to a wild-type cell of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) reduced endogenous B2M expression relative to wild-type cells of the same cell type; (iii) reduced endogenous CIITA expression relative to wild-type cells of the same cell type; and (iv) Fas-CD27. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-CD27. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) reduced expression of CIITA; and (iv) nucleic acid encoding Fas-CD27. In some aspects, the modified cell comprises: (i) CAR; (ii) reduced expression of endogenous B2M; (iii) reduced expression of CIITA; (iv) reduced expression of endogenous CD58 relative to wild-type cells of the same cell type; and (v) nucleic acid encoding Fas-CD27.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-CD27. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-CD27. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27.

In some aspects, the cell further comprises Fas-4-1BB, such as Fas-4-1BB disclosed herein. Thus, some aspects of the disclosure relate to a modified cell, such as an immune cell, comprising: (i) a CAR or an eTCR; (ii) an endogenous MHC-I human leukocyte antigen that is reduced in expression relative to a wild-type cell of the same cell type; (iii) an endogenous MHC-II human leukocyte antigen that is reduced in expression relative to a wild-type cell of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-4-1BB.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous CD58 whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-4-1BB.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-4-1BB. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-4-1BB.

In some aspects, the cell further comprises Fas-OX40, such as Fas-OX40 disclosed herein. Thus, some aspects of the disclosure relate to a modified cell, such as an immune cell, comprising: (i) a CAR or an eTCR; (ii) reduced expression of an endogenous MHC-I human leukocyte antigen relative to a wild-type cell of the same cell type; (iii) reduced expression of an endogenous MHC-II human leukocyte antigen relative to a wild-type cell of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cell, such as an immune cell, comprises: (i) a CAR or an eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-OX40.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-OX40. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-OX40. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous CD58 whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-OX40.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-OX40. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; and (iv) Fas-OX40. In some embodiments, the modified cell comprises: (i) eTCR; (ii) endogenous B2M expressed at a reduced level relative to wild-type cells of the same cell type; (iii) endogenous CIITA expressed at a reduced level relative to wild-type cells of the same cell type; (iv) endogenous CD58 expressed at a reduced level relative to wild-type cells of the same cell type; and (v) Fas-OX40. II.A.2. Improved expression of endogenous poliovirus receptor (PVR)

In some aspects, the modified cell further comprises reduced expression of an endogenous poliovirus receptor (PVR) relative to wild-type cells of the same cell type. In some aspects, reduced expression of PVR is caused by a mutation or deletion of one or more endogenous genes encoding PVR. In some aspects, the amount of PVR expressed 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 amount of PVR expressed in a wild-type cell of the same cell type. In some aspects, the cell has no detectable functional PVR expression. In some aspects, the cell has no detectable PVR expression.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-DN. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-DN. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-DN.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-DN. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-DN. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-DN.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-CD27. In some embodiments, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-CD27.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-CD27.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-4-1BB.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-4-1BB. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-4-1BB.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (v) Fas-OX40. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous B2M whose expression is reduced relative to wild-type cells of the same cell type; (iii) endogenous CIITA whose expression is reduced relative to wild-type cells of the same cell type; (iv) endogenous PVR whose expression is reduced relative to wild-type cells of the same cell type; and (v) Fas-OX40. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; and (vi) Fas-OX40.

In some aspects, the modified cell comprises: (i) eTCR; (ii) reduced expression of MHC-I HLA relative to wild-type cells of the same cell type; (iii) reduced expression of MHC-II HLA relative to wild-type cells of the same cell type; (iv) reduced expression of PVR; and (v) a nucleic acid encoding Fas-OX40. In some aspects, the modified cell comprises: (i) 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 cell comprises: (i) 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. II.A.3. Improved expression of HLA-E

In some aspects, the modified cell further comprises increased expression of HLA-E relative to a wild-type cell of the same cell type (e.g., a cell prior to modification). In some aspects, the increased expression of HLA-E results from transfection of a nucleic acid encoding an HLA-E polypeptide. In some aspects, 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 aspects, 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 aspects, the amount of HLA-E expressed is at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least 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 amount of HLA-E expressed in a wild-type cell of the same cell type.

In some aspects, the modified cell further comprises HLA- E. In some aspects, the amount of HLA-E expressed is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% of the amount of HLA-E expressed in a wild-type cell of the same cell type.

In some aspects, the HLA-E is a human HLA-E polypeptide. In some aspects, the HLA-E polypeptide is a chimeric polypeptide comprising a human HLA-E polypeptide or a portion thereof. In some aspects, the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a human B2M polypeptide. In some aspects, the HLA-E polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 32.

In some aspects, 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 shown in SEQ ID NO: 33, 36, or 38. In some aspects, HLA-E is encoded by the nucleotide sequence shown in SEQ ID NO: 33, 36, or 38.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (v) Fas-DN. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-DN. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-DN. In some embodiments, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (vi) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vii) Fas-DN.

In some aspects, the modified cell comprises: (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) increased expression of HLA-E relative to wild-type cells of the same cell type; and (v) Fas-DN. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-DN. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) a nucleic acid encoding Fas-DN. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (vi) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vii) Fas-DN.

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-CD27. In some embodiments, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-CD27. In some embodiments, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (vi) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vii) Fas-CD27.

In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (v) Fas-CD27. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-CD27. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-CD27. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (vi) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vii) Fas-CD27.

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

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

In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (v) Fas-OX40. In some aspects, the modified cell comprises: (i) CAR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-OX40. In some aspects, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is decreased in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is decreased in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is decreased in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-OX40. In some embodiments, the modified cells comprise: (i) CAR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous CD58 that is reduced in expression relative to wild-type cells of the same cell type; (v) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (vi) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vii) Fas-OX40.

In some aspects, the modified cell comprises: (i) an eTCR; (ii) an endogenous MHC-I HLA that is reduced in expression relative to a wild-type cell of the same cell type; (iii) an endogenous MHC-II HLA that is reduced in expression relative to a wild-type cell of the same cell type; (iv) an increased expression of HLA-E relative to a wild-type cell of the same cell type; and (v) Fas-OX40. In some aspects, the modified cell comprises: (i) eTCR; (ii) endogenous MHC-I HLA that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous MHC-II HLA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-OX40. In some aspects, the modified cells comprise: (i) eTCR; (ii) endogenous B2M that is reduced in expression relative to wild-type cells of the same cell type; (iii) endogenous CIITA that is reduced in expression relative to wild-type cells of the same cell type; (iv) endogenous PVR that is reduced in expression relative to wild-type cells of the same cell type; (v) HLA-E that is increased in expression relative to wild-type cells of the same cell type; and (vi) Fas-OX40. In some embodiments, the modified cell comprises: (i) eTCR; (ii) endogenous B2M expressed at a reduced level relative to wild-type cells of the same cell type; (iii) endogenous CIITA expressed at a reduced level relative to wild-type cells of the same cell type; (iv) endogenous CD58 expressed at a reduced level relative to wild-type cells of the same cell type; (v) endogenous PVR expressed at a reduced level relative to wild-type cells of the same cell type; (vi) HLA-E expressed at an increased level relative to wild-type cells of the same cell type; and (vii) Fas-OX40. II.A.4. Additional Polypeptides

In some aspects, the modified cell also overexpresses one or more additional endogenous polypeptides. In some aspects, the modified cell also expresses one or more additional heterologous polypeptides. In some aspects, the modified cell comprises a human interleukin 15 (IL15) polypeptide. In some aspects, the modified cell overexpresses an endogenous IL15 polypeptide. In some aspects, the modified cell expresses a heterologous IL15 polypeptide. In some aspects, the IL15 polypeptide is a membrane-bound IL15 polypeptide. In some aspects, the IL15 polypeptide is a membrane-bound IL15/IL15Rα fusion polypeptide (mIL15/Ra). In some aspects, the IL15 polypeptide comprises an IL15 sushi domain/IL15Ra fusion polypeptide (sushi15). In some aspects, the IL15 polypeptide comprises a membrane-bound IL15/IL15Ra-LSP fusion (mIL15/Ra-LSP). In some aspects, the IL15 polypeptide comprises a short IL15 polypeptide (sIL15). In some aspects, the IL15 polypeptide comprises a soluble IL15 polypeptide.

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

In some aspects, the modified cell comprises a HSVTK polypeptide. In some aspects, 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 aspects, the HSVTK polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 34, 36, or 39. II.A.5. Cells

Any cell type can be used in the compositions and methods disclosed herein. In some aspects, the cell is an immune cell. In some aspects, the cell is an induced pluripotent stem cell (iPSC). In some aspects, the cell is an embryonic stem cell (ESC). In some aspects, the cell is an immune cell selected from T cells, NK cells, NKT cells, or tumor infiltrating lymphocytes. In some aspects, the cell is a T cell. In some aspects, the cell is an α-β T cell or a γ-δ T cell. In some aspects, the cell is a γδ T cell comprising Vδ1. In some aspects, the cell is a γδ T cell comprising Vδ2.

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

In some aspects, the cell comprises a CAR or an eTCR, wherein the CAR or the eTCR comprises an antigen binding domain that specifically binds to a tumor antigen. In some aspects, the tumor antigen comprises CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-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, claudin (e.g., CLDN18.2), B7H3, or any combination thereof.

In some aspects, the cell comprises a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to mesothelin. In some aspects, the cell comprises an eTCR, wherein the eTCR comprises an antigen binding domain that specifically binds to mesothelin. In some aspects, the cell comprises an eTCR, wherein the eTCR is a gamma-delta TCR. In some aspects, the cell comprises an eTCR, wherein the eTCR is a g9d2 TCR.

In some aspects, the cell comprises a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to CD 19. In some aspects, the cell comprises an eTCR, wherein the eTCR comprises an antigen binding domain that specifically binds to CD19.

In some aspects, the cell comprises a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to BCMA. In some aspects, the cell comprises an eTCR, wherein the eTCR comprises an antigen binding domain that specifically binds to BCMA.

Some aspects of the disclosure relate to a cell population comprising one or more modified cells disclosed herein. In some aspects, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cells in the cell population comprise 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 in the cell population are modified cells disclosed herein. In some aspects, at least about 25% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 30% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 35% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 40% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 45% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 50% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 55% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 60% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 65% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 70% of the cells in the cell population comprise modified cells disclosed herein. In some aspects, at least about 75% of the cells in the cell population comprise modified cells disclosed herein. II.B. Fas Constructs

Some aspects of the present disclosure relate to modified polypeptides and chimeric polypeptides comprising the extracellular domain of Fas receptor. Other aspects relate to modified cells, such as immune cells, such as iCAR-T cells, which include a modified polypeptide or chimeric polypeptide containing the extracellular domain of Fas receptor (Fas ECD). II.B.1. Fas-DN

In some aspects, the modified polypeptide is a dominant negative Fas (Fas-DN). Therefore, some aspects of the present disclosure are related to a modified cell comprising Fas-DN, such as an immune cell, such as an iCAR-T cell. In some aspects, Fas-DN comprises a modification (e.g., a deletion or substitution) of one or more amino acids in the extracellular domain of the Fas receptor and the intracellular (cytoplasmic) domain of the Fas receptor. In some aspects, Fas-DN comprises a deletion of one or more amino acids in the extracellular domain of the Fas receptor and the intracellular domain of the Fas receptor. In some aspects, the modification (e.g., a deletion or substitution) is in one or more amino acids selected from residues 191-335 of the Fas sequence. In some aspects, Fas-DN has a reduced interaction with the Fas ligand or no interaction. In some aspects, Fas-DN is unable to recruit and/or activate caspase 8.

In some aspects, Fas-DN comprises the extracellular domain of Fas receptor and the truncated intracellular domain of Fas receptor. In some aspects, the truncated Fas receptor comprises a deletion of one or more amino acids selected from residues 230 to 312 of the canonical Fas receptor amino acid sequence (SEQ ID NO: 30). In some aspects, 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 to 312 of the classical Fas receptor amino acid sequence (SEQ ID NO: 30). In some aspects, 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 to 312 of the classical Fas receptor amino acid sequence (SEQ ID NO: 30), residues 230 to 305, residues 230 to 306, residues 230 to 307, residues 230 to 308, residues 230 to 309, residues 230 to 310, residues 230 to 311, residues 230 to 312, residues 231 to 312, residues 232 to 312, residues 233 to 312, residues 234 to 312, residues 235 to 312, residues 236 to 312, residues 237 to 312, residues 238 to 312, residues 239 to 312 or residues 240 to 312 of amino acid ... In some aspects, 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 one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten additional amino acid deletions. In some aspects, the truncated Fas receptor comprises a deletion of residues 230 to 313, residues 230 to 314, residues 230 to 315, residues 230 to 316, residues 230 to 317, residues 229 to 312, residues 228 to 312, residues 227 to 312, residues 226 to 312, or residues 225 to 312 of the canonical Fas receptor amino acid sequence (SEQ ID NO: 30). In some aspects, the truncated Fas receptor comprises a deletion of residues 230 to 312 of the canonical Fas receptor amino acid sequence (SEQ ID NO: 30).

In some aspects, 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 shown in SEQ ID NO: 22. In some aspects, the chimeric polypeptide comprises the amino acid sequence shown in SEQ ID NO: 27. In some aspects, Fas-DN comprises the amino acid sequence shown in SEQ ID NO: 27. In some aspects, Fas-DN further comprises a signal peptide. In some aspects, Fas-DN includes a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 28. In some aspects, Fas-DN includes a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 29. II.B.2. Fas-CD27

In some aspects, the chimeric polypeptide is a Fas-CD27 chimeric polypeptide (Fas-CD27). Thus, some aspects of the disclosure relate to a modified cell comprising Fas-CD27, such as an immune cell, such as an iCAR-T cell. In some aspects, the chimeric polypeptide comprises an extracellular domain of Fas linked to an intracellular domain of CD27 or a portion thereof. In some aspects, the chimeric polypeptide comprises an extracellular domain of Fas linked to a portion of an intracellular domain of CD27, wherein the portion of the intracellular domain of CD27 retains one or more intracellular signaling functions of full-length CD27.

In some aspects, the 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 shown in SEQ ID NO: 22. In some aspects, the Fas-CD27 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 22. In some aspects, Fas-CD27 further comprises a signal peptide. In some aspects, Fas-CD27 includes a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 28. In some aspects, Fas-CD27 includes a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 29. II.B.3. Fas-4-1BB

In some aspects, the chimeric polypeptide is a Fas-4-1BB chimeric polypeptide (Fas-4-1BB). Therefore, some aspects of the present disclosure are related to a modified cell comprising Fas-4-1BB, such as an immune cell, such as an iCAR-T cell. In some aspects, the chimeric polypeptide comprises a Fas extracellular domain connected to a 4-1BB intracellular domain or a portion thereof. In some aspects, the chimeric polypeptide comprises a Fas extracellular domain connected to a portion of a 4-1BB intracellular domain, wherein the portion of the 4-1BB intracellular domain retains one or more intracellular signaling functions of the full-length 4-1BB.

In some aspects, the 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 shown in SEQ ID NO: 23. In some aspects, the Fas-4-1BB polypeptide comprises the amino acid sequence shown in SEQ ID NO: 23. In some aspects, Fas-4-1BB further comprises a signal peptide. In some aspects, Fas-4-1BB includes a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 28. In some aspects, Fas-4-1BB includes a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 29. II.B.4. Fas-OX40

In some aspects, the chimeric polypeptide is a Fas-OX40 chimeric polypeptide (Fas-OX40). Thus, some aspects of the disclosure relate to a modified cell comprising Fas-OX40, such as an immune cell, such as an iCAR-T cell. In some aspects, the chimeric polypeptide comprises an extracellular domain of Fas linked to an intracellular domain of OX40 or a portion thereof. In some aspects, the chimeric polypeptide comprises an extracellular domain of Fas linked to a portion of an intracellular domain of OX40, wherein the portion of the intracellular domain of OX40 retains one or more intracellular signaling functions of the full-length OX40.

In some aspects, the 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 aspects, the chimeric polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24.

In some aspects, 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 aspects, the chimeric polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25.

In some aspects, 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 shown in SEQ ID NO: 26. In some aspects, the chimeric polypeptide comprises the amino acid sequence shown in SEQ ID NO: 26.

In some aspects, Fas-OX40 further comprises a signal peptide. In some aspects, Fas-OX40 comprises a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 28. In some aspects, Fas-OX40 comprises a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 29. II.C. CD58- specific guide RNA

Some aspects of the disclosure relate to a guide RNA capable of hybridizing with a human CD58 gene. In some aspects, the guide RNA hybridizes with a nucleotide sequence in exon 3 of an endogenous CD58 gene. In some aspects, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 1-20 and 41.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 1. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 1. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 1.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 2. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 2. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 2.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 3. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 3. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 3.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 4. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 4. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 4.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 5. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 5. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 5.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 6. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 6. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 6.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 7. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 7. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 7.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 8. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 8. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 8.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 9. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 9. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 9.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 10. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 10. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 10.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 11. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 11. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 11.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 12. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 12. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 12.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 13. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 13. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 13.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 14. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 14. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 14.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 15. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 15. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 15.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 16. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 16. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 16.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 17. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 17. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 17.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 18. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 18. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 18.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 19. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 19. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 19.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 20. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 20. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 20.

In some aspects, the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 41. In some aspects, the guide RNA consists of the nucleotide sequence shown in SEQ ID NO: 41. In some aspects, the guide RNA comprises a nucleotide sequence hybridized with a nucleotide sequence overlapping with a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 41.

Some aspects of the present disclosure are related to a kit comprising (i) a guide RNA disclosed herein capable of hybridizing with a human CD58 gene and (ii) a gene editing tool. In some aspects, the gene editing tool includes CRISPR/Cas9, CRISPR/Cas12, TALEN, zinc finger nuclease, or any combination thereof. In some aspects, the kit comprises (i) a guide RNA comprising a nucleotide sequence shown in SEQ ID NO: 1, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising a nucleotide sequence shown in SEQ ID NO: 2, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising a nucleotide sequence shown in SEQ ID NO: 3, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising a nucleotide sequence shown in SEQ ID NO: 4, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO:5, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO:6, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO:7, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO:8, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO:9, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO:10, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 11, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 12, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 13, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 14, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 15, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 19, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 17, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 18, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 19, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 20, and (ii) CRISPR/Cas9. In some aspects, the kit comprises (i) a guide RNA comprising the nucleotide sequence shown in SEQ ID NO: 41, and (ii) CRISPR/Cas9. III. Methods of the Disclosure III.A. Engineering Methods

Some aspects of the present disclosure relate to methods of engineering human cells, comprising: (i) transfecting the cells with a nucleic acid encoding a CAR or an eTCR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting the cells with a nucleic acid encoding a polypeptide comprising a Fas-DN, a Fas-CD27 chimeric polypeptide, a Fas-4-1BB chimeric polypeptide, a Fas-OX40 chimeric polypeptide, or any combination thereof.

Other aspects of the disclosure relate to methods for engineering human cells, comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of MHC-I human leukocyte antigens relative 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 reduced expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iii) transfecting the cell 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.

Other aspects of the disclosure relate to methods of engineering human cells comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of MHC-I human leukocyte antigens relative 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 reduced expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iii) inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in reduced expression of CD58 relative to wild-type cells of the same cell type.

Other aspects of the disclosure relate to methods for engineering human cells, comprising: (i) inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of the MHC-I human leukocyte antigens relative 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 reduced expression of the MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; (iii) inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in reduced expression of CD58 relative to wild-type cells of the same cell type; and (iv) inactivating one or more endogenous genes encoding a dominant negative Fas The cell is transfected with a nucleic acid encoding a Fas-DN (Fas-DN), a nucleic acid encoding an exogenous HLA-E polypeptide, a nucleic acid encoding a suicide gene, or any combination thereof.

In some aspects, the CAR or eTCR comprises an antigen binding domain that specifically binds to a tumor antigen. In some aspects, the tumor antigen comprises CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-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, claudin (e.g., CLDN18.2), B7H3, or any combination thereof.

In some aspects, the method comprises: (i) transfecting cells with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting cells with a nucleic acid encoding a polypeptide comprising a Fas-DN, a Fas-CD27 chimeric polypeptide, a Fas-4-1BB chimeric polypeptide, a Fas-OX40 chimeric polypeptide, or any combination thereof.

In some aspects, 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 mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-DN, a Fas-CD27 chimeric polypeptide, a Fas-4-1BB chimeric polypeptide, a Fas-OX40 chimeric polypeptide, or any combination thereof.

In some aspects, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative 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 aspects, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-CD27 chimeric polypeptide (e.g., disclosed herein). In some aspects, the method comprises: (i) transfecting cells with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting cells with a nucleic acid encoding a polypeptide comprising a Fas-4-1BB chimeric polypeptide (e.g., disclosed herein). In some aspects, the method comprises: (i) transfecting a cell with a nucleic acid encoding a CAR; (ii) mutating or deleting one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-OX40 chimeric polypeptide (e.g., disclosed herein).

In some aspects, 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 mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative 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 aspects, 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 mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-CD27 chimeric polypeptide (e.g., disclosed herein). In some aspects, 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 mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-4-1BB chimeric polypeptide (e.g., disclosed herein). In some aspects, 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 mutation or deletion reduces the expression of MHC-I human leukocyte antigens relative 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 mutation or deletion reduces the expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-OX40 chimeric polypeptide, such as 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 B2M, wherein the mutation or deletion reduces the expression of B2M relative to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion reduces the expression of MHC-II human leukocyte antigen relative 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 B2M, wherein the mutation or deletion reduces the expression of B2M relative to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion reduces the expression of MHC-II human leukocyte antigen relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-CD27 chimeric polypeptide (e.g., 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 B2M, wherein the mutation or deletion reduces the expression of B2M relative to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion reduces the expression of MHC-II human leukocyte antigen relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-4-1BB chimeric polypeptide (e.g., disclosed herein).

In some aspects, 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 reduces the expression of B2M relative to wild-type cells of the same cell type; (iii) mutating or deleting CIITA, wherein the mutation or deletion reduces the expression of MHC-II human leukocyte antigen relative to wild-type cells of the same cell type; and (iv) transfecting a cell with a nucleic acid encoding a polypeptide comprising a Fas-OX40 chimeric polypeptide (e.g., disclosed herein).

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

In some aspects, the method further comprises mutating or deleting one or more endogenous genes encoding PVR, wherein the mutation or deletion reduces the expression of PVR relative to wild-type cells 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 using gene editing tools, such as those disclosed herein.

In some aspects, 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 aspects, the method further comprises modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide. In some aspects, the method further comprises (i) transfecting the cell with a nucleic acid encoding an HLA-E polypeptide; and (ii) modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide. In some aspects, the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a B2M polypeptide.

In some aspects, the method further comprises transfecting the cell with a nucleic acid encoding an IL15 polypeptide, such as an IL15 polypeptide disclosed herein. In some aspects, the IL15 polypeptide is a membrane-bound IL15 polypeptide. In some aspects, the IL15 polypeptide is a membrane-bound IL15/IL15Rα fusion polypeptide (mIL15/Ra). In some aspects, the IL15 polypeptide comprises an IL15 sushi domain/IL15Ra fusion polypeptide (sushi15). In some aspects, the IL15 polypeptide comprises a membrane-bound IL15/IL15Ra-LSP fusion (mIL15/Ra-LSP). In some aspects, the IL15 polypeptide comprises a short IL15 polypeptide (sIL15). In some aspects, the IL15 polypeptide comprises a soluble IL15 polypeptide.

In some aspects, the method further comprises transfecting the cell with a nucleic acid encoding a CCL19 polypeptide.

In some aspects, the endogenous gene can be inactivated by inserting an exogenous gene into the site of the endogenous gene. In some aspects, 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.

In some aspects, the promoter is a CAG promoter, an EF1α promoter, a CMV promoter, an hPGK promoter, or any combination thereof. In some aspects, the promoter comprises the nucleotide sequence shown in SEQ ID NO: 37.

In some aspects, the tagged protein is LNGFR or a truncated LNGFR (e.g., delta LNGFR). In some aspects, the tagged protein is EGFR. In some aspects, the tagged protein is a truncated EGFR (tEGFR).

In some embodiments, the exogenous gene intended to be inserted into the endogenous gene site comprises HLA-E and CAG promoter. In some embodiments, the exogenous gene intended to be inserted into the endogenous gene site comprises FasDN and CAG promoter. In some embodiments, the exogenous gene intended to be inserted into the endogenous gene site comprises HLA-E, HSV-TK, FasDN and CAG promoter. In some embodiments, the exogenous gene intended to be inserted into the endogenous gene site comprises HLA-E, HSV-TK, FasDN, EF1α promoter and CAG promoter. In some embodiments, the exogenous gene intended to be inserted into the endogenous gene site comprises HLA-E, HSV-TK, FasDN, LNGFR or CAG promoter.

In some embodiments, the exogenous gene is inserted into the site of the B2M gene. In some embodiments, the exogenous gene is inserted into the site of the CIITA gene. In some embodiments, the exogenous gene is inserted into the site of the CD58 gene.

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

In some embodiments, the cell is an iPSC. In some embodiments, the cell is a cell differentiated from an iPSC. In some embodiments, the cell is an immune cell differentiated from an iPSC. In some embodiments, the cell is a T cell differentiated from an iPSC. In some embodiments, the cell is an NK cell differentiated from an iPSC. In some embodiments, the cell is an NKT cell differentiated from an iPSC. When the cell is an iPSC, it can be used not only as a source of immune cells, but also as a source of any other type of cell that can be differentiated from an iPSC. Such differentiated cells have reduced in vivo immunogenicity and can be used in any cell therapy including administering or transplanting such cells to a patient. III.B. Methods of Treatment

Some aspects of the disclosure are directed to methods of treating a disease or condition in an individual in need thereof, comprising administering to the individual a composition disclosed herein. In some aspects, the method comprises administering a modified or engineered cell disclosed herein. In some aspects, the method comprises administering a cell population disclosed herein. In some aspects, the method comprises administering a composition comprising a Fas construct disclosed herein. In some aspects, the method comprises administering a composition comprising a CD58-specific guide RNA.

In some aspects, the disease or disorder comprises cancer, e.g., the individual suffers from cancer. In some aspects, cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or eye, 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, In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is recurrent. In some aspects, the cancer is refractory or recurrent after one or more prior anticancer therapies. In some aspects, the one or more prior anticancer therapies comprise standard of care therapy.

In some aspects, the compositions disclosed herein are administered in combination with another anticancer therapy. In some aspects, the other anticancer therapy comprises chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof. In some aspects, the other anticancer therapy comprises chemotherapy. In some aspects, the other anticancer therapy comprises an immune checkpoint inhibitor. In some aspects, the other anticancer 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 aspects, the anticancer therapy comprises an antibody or an antigen-binding portion thereof that specifically binds and inhibits PD-1. In some aspects, the anti-cancer therapy comprises an antibody, or an antigen-binding portion thereof, that specifically binds and inhibits PD-L1.

In some embodiments, the method further comprises pre-treating the individual prior to administering the immune cell population. In some embodiments, chemotherapy is administered to the individual prior to administering the immune cell population. In some embodiments, immunodepleting chemotherapy is administered to the individual prior to administering the immune cell population. In some embodiments, the immunodepleting chemotherapy comprises cyclophosphamide, fludarabine, or both.

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

In some aspects, the cells of the present disclosure are at least about 1×10 ⁶ cells, at least about 2×10 ⁶ cells, at least about 3×10 ⁶ cells, at least about 4×10 ⁶ cells, at least about 5×10 ⁶ cells, 1×10 ⁷ cells, at least about 2×10 ⁷ cells, at least about 3×10 ⁷ cells, at least about 4×10 ⁷ cells, at least about 5×10 ⁷ cells, 1×10 ⁸ cells, at least about 2×10 ⁸ cells, at least about 3×10 ⁸ cells, at least about 4×10 ⁸ cells, at least about 5×10 ⁸ cells, 1×10 ⁹ cells, at least about 2×10 ⁹ cells, at least about 3×10 ⁹ cells, at least about 4×10 ⁹ cells, or at least about 5×10 ⁹ cells. III.C. Cell Therapy Products

Some aspects of the disclosure are related to a cell population comprising a plurality of modified cells disclosed herein. Some aspects of the disclosure are related to a cell therapy method comprising a modified cell population disclosed herein. In some aspects, the cell population is cryopreserved. Any cryopreservation method of 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 method is cryopreserved to facilitate the transportation of the cells.

The cell therapies and/or cell populations 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 aspects, the cell therapies and/or cell populations are formulated with one or more excipients that allow cryopreservation of cells, such as DMSO.

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

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD19. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD19. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD20. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD20. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD20.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 ROR1. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD22. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD22. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 a carcinoembryonic antigen. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 alpha-fetoprotein. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 alpha-fetoprotein.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CA-125. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CA-125.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 5T4. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 MUC-1. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 an epithelial tumor antigen. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (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 an epithelial tumor antigen. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 an epithelial tumor antigen.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 a prostate-specific antigen. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 a melanoma-associated antigen. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 a melanoma-associated antigen. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 a melanoma-associated antigen.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 mutant p53. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 mutant p53. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 mutant p53.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 mutant ras. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 a mutant ras. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 a mutant ras.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 HER2/Neu. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 a folate binding protein. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 folate binding protein.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type or relative to cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type or relative to cells before modification; (iii) Fas (Fas-DN), increased expression of HLA-E relative to wild-type cells of the same cell type or relative to cells before modification, 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 HIV-1 envelope glycoprotein gpl20. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 HIV-1 envelope glycoprotein gpl20. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 HIV-1 envelope glycoprotein gpl20.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type or relative to cells before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type or relative to cells before modification; (iii) Fas (Fas-DN), increased expression of HLA-E relative to wild-type cells of the same cell type or relative to cells before modification, 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 HIV-1 envelope glycoprotein gp41. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 HIV-1 envelope glycoprotein gp41. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 HIV-1 envelope glycoprotein gp41.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 GD2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 GD2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 GD2.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD123. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD123. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD123.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD33. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD33. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD138. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD138. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD138.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD23. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD23. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD23.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD30. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD30. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD30.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CD56. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD56. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CD56.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 c-Met. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 c-Met. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 c-Met.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 GD3. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 GD3. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 GD3.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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-11Rα. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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-11Rα. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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-11Rα.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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-κ chain. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 a kappa chain. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 a kappa chain.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 lambda chain. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 CSPG4. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CSPG4. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 CSPG4.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 ERBB2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 ERBB2.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 a HER2-HER3 combination. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 HER2-HER3 combination. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 HER2-HER3 combination.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 HER1-HER2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 NY-ESO-1. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 NY-ESO-1. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 NY-ESO-1.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 SSX2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 SSX2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 SSX2.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 MAGE. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 MAGE.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 MART-1. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 gp100. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 gp100. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 gp100.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 PSA. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 PSMA. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 PSMA. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 PSMA.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell prior to modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell prior to modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell prior to 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 PSCA. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 PSCA. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 PSCA.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 GPC3. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 EpCAM. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, the plurality of modified cells comprising: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 BCMA.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 GCC. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 GCC. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 GCC.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 ADGRE2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 ADGRE2. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 ADGRE2.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 claudin. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 claudin. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 claudin.

Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (ii) reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to a wild-type cell of the same cell type or relative to a cell before modification; (iii) increased expression of Fas (Fas-DN), HLA-E relative to a wild-type cell of the same cell type or relative to a cell 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 B7H3. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 B7H3. Some aspects of the present disclosure relate to a cell population comprising a plurality of modified cells, wherein the plurality of modified cells comprises: (i) an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii) an inactivated endogenous gene encoding 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 B7H3.

Unless otherwise indicated, the practice of the present disclosure will employ techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully described in the literature. See, e.g., Sambrook et al., eds. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover, ed. (1985) DNA Cloning, Vols. I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular 1986); Crooks, Antisense drug Technology: Principles, strategies and applications, 2nd edition CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, MD). Md.).

All references cited above and all references cited herein are incorporated by reference in their entirety.

The following examples are provided by way of illustration and not limitation. Examples Example 1 Isolation and expansion of peripheral blood mononuclear cell-derived alloreactive T cells (allo-T cells )

T cells were negatively selected from peripheral blood mononuclear cells (PBMCs) by immunomagnetic separation using the EasySep Human T Cell Isolation Kit (Stemcell Technologies). Anti-mesothelin (MESO) chimeric antigen receptor (iCAR) T cells produced by modified T cells derived from induced pluripotent cells (iPCs) were irradiated with 25 Gy for 4 cycles. Then, meso iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) for 30 minutes at 37°C in the dark. The labeling reaction was quenched with 40 mL of complete medium. Then, the irradiated meso iCAR-T cells were co-cultured with T cells in the presence of 2 ng/mL IL-2 for 10 days. The medium was changed on the 4th and 7th days of culture. Then, the alloreactive T (allo-T) cells were stored and frozen. In vitro alloreactive T cell (allo-T cell ) activation and cytotoxicity analysis

Allo-T cells were co-cultured with meso iCAR-T cells to evaluate the killing effect of allo-T cells on meso iCAR-T cells. Wild-type (WT) meso iCAR-T cells and MHC-I and MHC-II double knockout (dKO) meso iCAR-T cells were labeled with cell trace violet (CTV; Life Technologies) stain for 30 minutes in the dark at 37°C. Approximately 1×10 ⁴ CTV _- stained meso iCAR-T cells were co-cultured with HLA-matched or HLA-mismatched allo-T cells at effector:target (E:T) ratios of 4:1, 2:1, 1:1, and 0:1 in a humidified 5% CO 2 incubator at 37°C for 24 hours in a 96-well plate. The co-cultured cells were centrifuged at 350 g for 5 minutes and then washed with PBS. The cells were then incubated with a fixable live/dead stain at room temperature for 20 minutes. After incubation, the cells were then washed with FACS buffer.

Co-cultured allo-T cells and WT or dKO meso iCAR-T cells were stained with anti-CTV and anti-CD25, operated on a FACS Canto and analyzed using FlowJo software. The activation state of allo-T cells was determined by detecting the CD25 activation marker (Figure 1A). Allo-T cells were activated and showed CD25 expression in PBMCs exposed to HLA-mismatched wild-type iCAR-T cells (Figure 1A), but not in PBMCs exposed to HLA-matched iCAR-T cells (Figure 1C). In contrast, PBMCs cultured with MHC-I/MHC-II dKO meso iCAR-T cells did not cause activation of allo-T cells (Figures 1B and 1C). Quantification of these representative graphs is shown in Figure 1E. Cytotoxicity of allo-T cells in vitro

Expanded and stimulated allo-T cells were co-cultured with meso iCAR-T cells to assess allo-T cell-mediated killing of meso iCAR-T cells. Four different meso iCAR-T cell lines were used in this experiment: WT, β2M/CTIIA double KO, HLA-ABC/CTIIA double KO, and HLA-ABC/CIITA double KO overexpressing HLA-G and HLA-E. Meso iCAR-T cells were labeled with cell trace violet (CTV; Life Technologies) stain for 30 minutes at 37°C in the dark. About 1×10 ⁴ CTV-stained meso iCAR-T cells were co-cultured with HLA-matched or HLA-mismatched allo-T cells at an effector:target (E: _T ) ratio of 2:1, 1:1 in a 96-well plate in a humidified 5% CO 2 incubator at 37°C for 24 hours. The co-cultured cells were centrifuged at 350 × g for 5 minutes and then washed with PBS. The cells were then incubated with a fixable live/dead cell stain at room temperature for 20 minutes. After incubation, the cells were then washed with flow cytometry (FACS) buffer. Then, flow cytometry was performed on the cells. Hypoimmunoedited meso iCAR-T cells, including β2M/CTIIA double KO, HLA-ABC/CTIIA double KO, or HLA-ABC/CIITA double KO, and overexpressing HLA-G and HLA-E, had reduced recognition and killing of mismatched allo-T cells (Figure 2). In vitro NK cell cytotoxicity assay

Natural killer (NK) cells from four different donors were co-cultured with meso iCAR-T cells to evaluate the killing effect of NK cells on meso iCAR-T cells. Meso iCAR-T cells were then labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) for 30 minutes at 37°C in the dark. The labeling reaction was quenched with 40 mL of complete medium. Approximately 1×10 ⁴ CFSE-stained meso iCAR-T cells were co-cultured with NK cells at effector:target (E: _T ) ratios of 5:1, 2:1, 1:1, and 0:1 in a humidified 5% CO 2 incubator at 37°C for 24 hours in a 96-well plate. The co-cultured cells were centrifuged at 350 g for 5 minutes and then washed with PBS. The cells were then incubated with a fixable live/dead stain at room temperature for 20 minutes. After incubation, the cells were then washed with FACS buffer.

NK cell cytotoxicity analysis was performed on different groups of hypoimmunoedited meso iCAR-T cells including B2M/CTIIA double KO (DKO) (Fig. 3A, Fig. 3E, Fig. 3I, and Fig. 3M); DKO and PVR KO (Fig. 3B, Fig. 3F, Fig. 3J, and Fig. 3N); DKO and CD58 KO (Fig. 3C, Fig. 3G, Fig. 3K, and Fig. 3O); and DKO, CD58 KO, and PVR KO (Fig. 3D, Fig. 3H, Fig. 3L, and Fig. 3P) at iCAR-T effector:target (E:T) ratios of 5:1, 2:1, and 0:1 for four different donors. These cells were compared to WT, non-transduced HLA-E (OE) overexpressing, and FasDN OE or HLA-E OE meso iCAR-T cells.

NK cell cytotoxicity analysis was performed on the following hypoimmunoedited meso iCAR-T cells at E:T ratios of 4:1, 2:1, and 1:1: B2M/CTIIA double KO (DKO); DKO and CD58 KO (TKO); TKO and overexpressing HLA-E and FasDN; TKO and overexpressing HLA-E and HSVTK; TKO and overexpressing HLA-E, FasDN, and HSVTK; and TKO and overexpressing HLA-E, FasBB, and HSVTK (Figure 4). NK cell cytotoxicity assays were performed on different groups of hypoimmunoedited meso iCAR-Ts at effector:target (E:T) ratios of 5:1 and 1.5:1, including WT-Thy1.1, WT-CD47, WT-FasDN, WT-CD47-FasDN, B2M-Thy1.1, B2M-CD47, B2M-FasDN, and B2M-CD47-FasDN. NK cells were derived from different donors (Figure 5A to Figure 5C). In vitro cytotoxicity assays of tumor cells

Meso iCAR-T cells were co-cultured with human-derived gastric cancer (GSU) cells with high mesothelin expression levels to evaluate the killing effect of meso iCAR-T cells on GSU cancer cells. Meso iCAR-T cells were co-cultured with GSU cells in 96-well plates at effector:target (E:T) ratios of 10:1, 3:1, 1:1, and 0.3:1 in a humidified 5% CO ₂ incubator at 37°C for 24 hours. The co-cultured cells were then evaluated using the cell titer-glo luminescent viability assay to determine the number of viable cells based on ATP quantification. The co-cultures were incubated with the cell titer-glo reagent for 15 minutes, causing cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. Then, the luminescence signal was measured using a Pherastar plate reader. WT cells and low immunoedited cells showed no difference in the effector function of meso iCAR-T cells against GSU cancer cells (Figure 6). Example 2 Reporter Description

Induced pluripotent stem cells are transduced with a CAR containing firefly luciferase (Luc2). This reporter reacts with the substrate d-luciferin and produces a luminescent signal that can be observed between 600-800 nm (the optimal wavelength for bioluminescent imaging). VIVOGLO™ Luminescent Preparation

VIVOGLO™ fluorescein (Promega, catalog number P1043) was reconstituted in sterile DPBS (Gibco) at a concentration of 15 mg/mL and filtered through a 0.2 uM filter. The resulting sterile fluorescein was maintained at -20°C. On the day of the study, an aliquot of fluorescein was removed from -20°C storage, covered with foil to prevent light-driven degradation, and maintained at room temperature. It was then transferred to a vivarium for injection. NK Killing and Allo-T Cell Rejection — Mouse Background

Female NSG-MHC I/II DKO mutant mice aged 5 to 8 weeks are engineered to display a combination of severe combined immunodeficiency mutations ( scid ), IL-2 receptor gamma chain deficiency, class I MHC deficiency (H2-K and D), class II MHC deficiency (IA), and resistance to graft-versus-host disease (GVHD). This mouse strain provides a model that can be used to study the mechanisms of xenogeneic GVHD in vivo and to rapidly evaluate therapeutic agents. GSULuc Efficacy Analysis — Mouse Background

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice aged 5 to 8 weeks were used in this experiment. These mice carry two mutations on a NOD/ShiLtJ genetic background: severe combined immunodeficiency (scid) and a complete null allele of the IL-2 receptor common gamma chain (IL-2rg ^null ). The scid mutation occurs in the DNA repair complex protein Prkdc and renders the mouse deficient in B cells and T cells. The IL-2rg ^null mutation prevents interleukin signaling through multiple receptors, thereby causing a lack of functional NK cells. Severe immunodeficiency allows mice to be humanized by engraftment of human CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), patient-derived xenografts (PDX), or adult stem cells and tissues. Immunodeficient NSG mice enable studies of human immune function, infectious diseases, diabetes, oncology, and stem cell biology. In vitro NK cell cytotoxicity assay

On day 0, female NSG-MHC I/II DKO mice aged 5 to 8 weeks were weighed. 200 uL of freshly prepared NK cells resuspended in PBS were administered intraperitoneally to each mouse. One hour later, 200 uL of meso iCAR-T cells were administered intraperitoneally. One hour after administration of iCAR-T cells, 200 uL of d-luciferin substrate was injected intraperitoneally. 9 minutes later, the animals were anesthetized and their ventral surfaces were imaged on the IVIS Spectrum to obtain bioluminescent signals. The signal was then quantified as the total flux (photons/second) within the region of interest (ROI). This process was repeated on days 1, 3, and 6. The line graph shows the fold change over time. Fold changes were defined as the change in bioluminescent signal relative to baseline for each animal and are expressed as +/- SEM.

On day 0, 2.0×10 ⁶ NK cells resuspended in DPBS were administered to NSG-MHC I/II DKO animals 1 hour before administration of meso iCAR-T cells. After administration of NK cells, iCAR-T cells were prepared and resuspended in DPBS. Approximately 1 hour after NK cell administration, WT (positive control) and low immune meso iCAR-T cells were administered to each animal at a dose of 1×10 ⁶ cells, which were B2M/CIITA double KO (untransduced or UTD) (negative control); B2M/CIITA double KO and overexpressing HLA-E and FasDN; B2M/CIITA/PVR triple KO and overexpressing HLA-E and FasDN; B2M/CIITA/CD58 triple KO and overexpressing HLA-E and FasDN; and B2M/CIITA/CD58/PVR quadruple KO and overexpressing HLA-E and FasDN. WT is defined as induced pluripotent stem cells transduced with mesothelin CAR. The normalized time course (Fig. 7A) and the time course using cells from two different donors (Fig. 7B-C) included the addition of HLA-E and FasDN overexpression in both B2M/CIITA/CD58 KO and B2M/CIITA/PVR/CD58 KO.

An in vivo NK cell cytotoxicity assay was performed using meso iCAR-T cells overexpressing FasBB and the kill switch HSVTK to assess the effect on persistence. The assay was performed on the following hypoimmunoedited meso iCAR-T cells: B2M/CTIIA double KO (DKO); DKO and CD58 KO (TKO); TKO and overexpressing HLA-E and FasDN; TKO and overexpressing HLA-E and HSVTK; TKO and overexpressing HLA-E, FasDN, and HSVTK; and TKO and overexpressing HLA-E, FasBB, and HSVTK (Figure 8A). Alloreactive T (Allo-T) Cell Rejection Assay

On day 0, female NSG-MHC I/II DKO mice aged 5 to 8 weeks were weighed and administered ip with 200 uL of freshly prepared alloreactive T cells resuspended in PBS. One hour later, 200 uL of meso iCAR-T cells were administered ip to each of WT; DKO-UTD; DKO and overexpressing HLA-E and FASDN; DKO and PVR KO and overexpressing HLA-E and FASDN; DKO and CD58 KO and overexpressing HLA-E and FASDN; and DKO and CD58/PVR KO and overexpressing HLA-E and FasDN. Approximately 1 hour after administration of iCAR T cells, 200 uL of d-luciferin substrate was injected ip. After 9 minutes, the animals were anesthetized and their ventral surfaces were imaged on an IVIS Spectrum to obtain bioluminescent signals. The signals were then quantified as total flux (photons/second) within the region of interest (ROI). The process was repeated on days 1, 3, and 6 (Figure 8B). GSU Efficacy Model

Female NSG mice aged 5 to 8 weeks were administered ip with 200 uL of freshly prepared 1×10 ⁶ GSULuc cells resuspended in DPBS:Matrigel (1:1). On day 0, 200 uL of 1×10 ⁶ iCAR T cells WT; DKO; DKO and overexpressing HLA-E1 and FasDN; and DKO and CD58 KO and overexpressing HLA-E1 and FasDN; and negative control (PBS) were administered ip. On days 7, 14, 21, 28, 35, and 42, animals were imaged as described above (Figures 8C to 8D). Example 3 Experimental design for FAS switch receptors

Meso iCAR-T cells were plated at 10,000 cells/well in 96-well plates. Four cell lines were used in these experiments, including Meso iCAR-T cells with mIL15/Ra (5BB), dominant negative Fas (dnFAS), Fas-BB (FAS-4-1BB), and Fas-Ox40 (FAS-OX40) (Figures 9A to 9B). 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml Super Fas ligand or PBS (negative control) were added for 5 days. After 5 days, cells were labeled with cell tracer violet dye for live/dead cell staining and counted using flow cytometry. Fas-Ox40 showed increased cell proliferation after addition of Super Fas ligand, and the fold expansion of cells after activation with Fas-Ox40 was higher compared to all other cell lines (Figure 10A to Figure 10F). Long-term in vitro co-culture workflow

On the morning of the stimulation day, GSU target cells were plated at 40,000 cells/well. The following effector cells were used for this analysis: untransduced cells prepared with boost and non-boost protocols (UTD, UTD-boost); meso-iCART cells with 5BB prepared with boost and non-boost protocols (5BB-boost, 5BB); meso-iCART cells with 5BB and FasDN; and meso-iCART cells with 5BB and Fas-Ox40. In the boost protocol, frozen cells were allowed to recover for 3 days in IMDM medium containing 15% FBS and IL2, IL7, and IL15. Next, cells were activated in medium containing IL-2, IL-7, IL-15, IL-18, IL-21, Z-VAD inhibitor, and CD30 agonist antibodies on CD3/Retronectin coated plates. Cells were then transferred to GREX flasks on day 3 and harvested and stored on day 6. 100,000 cells/well of effector cells were added at an E:T ratio of 2.5:1 and then co-cultured for 48 hours. After 48 hours, effector cells were harvested, and effector cell proliferation was determined by counting live CD3+ cells using flow cytometry (Figure 11A), and the percentage of target cell lysis was analyzed by cell titer glo analysis (Figure 11B). At the same time, 200 uL of the collected effector cells were added to the fresh target cell plate seeded at 40,000 cells/well on the morning of stimulation and then co-cultured for 48 hours. This process was repeated for 2 weeks. At the end of 48 hours, the effector cells were analyzed using the method described above. In vivo GSU efficacy model

Female NSG mice aged 8 to 10 weeks were administered 100 uL of 1×10 ⁶ freshly prepared GSULuc cells resuspended in DPBS:Matrigel (1:1) intraperitoneally. On day 3, mice were randomized into groups and injected with meso-iCAR-T cells on day 4. Group 1 was administered PBS as a negative control. Treated mice were administered with untransduced mIL15/Ra (IL-15 or 5BB in Figures 12A-12B); 5BB and LNGFR; 5BB and FasDN; 5BB and Fas-41BB; 5BB and Fas-CD27; or 5BB and Fas-Ox40 (Figure 12A). The Fas-Ox40 group showed the highest efficacy, which is highlighted in Figures 12B and 12C. Example 4 Modification of iPS cells

iPS cells were modified by electroporation (EP). Ribonucleoprotein (RNP) complexes were generated by mixing Cas9 and gRNAs for B2M exon 1, CIITA exon 3, and CD58 exon 3 sites for gene knockout (KO). After RNP formation, plasmids of 1) HLA-E, 2) FasDN, and 3) HSVTK-tEGFR were constructed, respectively, with homology arms corresponding to each KI site generated by gene KO in the above procedure and CAG promoter for expression of each protein, and then the resulting solution was mixed with iPSCs resuspended in buffer for gene knock-in (KI). The KO/KI combinations of interest were as follows: 1) B2M KO/CAG-HLA-E KI; and 2) CIITA KO/CAG-FASDN KI, or 3) CIITA KO/CAG-HSVTK-tEGFR KI, or 4) B2M KO/CAG-HLA-E-HSVTK KI, and 5) CD58 KO. EP was performed and cells were then plated in 6-well plates. Two weeks after EP, modified cells were enriched for KI surface markers by magnetic bead sorting. One week after sorting, the percentage of KI population in modified cells was assessed using flow cytometry. Modification of iT or iCART cells

iT or iCART cells were modified by electroporation (EP). Ribonucleoprotein (RNP) complexes were generated by sequentially or simultaneously mixing Cas9 and gRNAs for B2M, CIITA, CD58, PVR, or HLA-ABC loci for gene knockout (KO). After RNP formation, EP was performed and cells were plated in 6-well plates for recovery culture. For KI, edited KO iT or iCART cells were transduced with gamma-retrovirus containing HLA-E or HLA-E-FASDN with and without HSVTK or HLA-G and with and without CD47 containing HLA-E or FASDN. Modified cells were enriched for KO/KI surface markers by magnetic bead sorting. One to two weeks after sorting, flow cytometry was used to assess the percentage of KO/KI populations of modified cells. Flow cytometry

iPSCs modified with 1) B2M KO/HLA-E KI or 2) CIITA KO/FasDN KI or 3) CIITA KO/CAG-HSVTK-tEGFR KI were evaluated by flow cytometry to identify populations expressing cell surface KI proteins. Cells were stained with anti-HLA-E, anti-Fas (CD95), or anti-tEGFR antibodies and Zombie Aqua Live/Dead Stain and evaluated by flow cytometry using the Attune NxT cytometer and flowjo analysis software. The efficacy of the KO/KI regimen for each KO/KI pair was determined independently by examining the percentage of viable cell populations positive for HLA-E in gene-edited cells (Figure 13A) compared to wild-type (WT) cells (Figure 13B), the percentage of viable cell populations positive for FasDN in gene-edited cells (Figure 13C) compared to WT cells (Figure 13D), and the percentage of viable cell populations positive for tEGFR in gene-edited cells (Figure 13E) compared to WT cells (Figure 13F). Gene-edited cells for HLA-E (Figure 13A), FasDN (Figure 13C), and HSVTK-tEGFR (Figure 13E) show successful KI of the genes of interest and subsequent expression. Example 5 CD58 KO

The KO efficiency of twenty CD58 gRNAs was evaluated in iCAR-T cells. By mixing appropriate amounts of Cas9 and CD58 gRNA, ribonucleoprotein (RNP) complexes ranging from 10 pmol to 100 pmol were produced. Subsequently, the resulting solution was mixed with iCAR-T cells resuspended in buffer for gene knockout (KO). Electroporation was performed, and after electroporation, the modified iCAR-T cells were plated in a 24-well plate. 7 to 10 days after electroporation, the CD58 knockout percentage was evaluated using flow cytometry. The KO efficiency is shown in Table 4: A: >75%, B: 65-75%, C: 50-65%, D: 30-50%, E: <30%. Table 4: CD58 KO efficiency CD58 guide RNA sequence KO efficiency SEQ ID NO: 1 AGGACTTATAATGTACTCAT B SEQ ID NO: 2 GTACTCATGGGATTGTCCTA B SEQ ID NO: 3 AGTCAATGCACAAGTTAGTG C SEQ ID NO: 4 AGTACATTATAAGTCCCTCGA C SEQ ID NO: 5 GCACAAGTTAGTGTGGGAGA A SEQ ID NO: 6 ACAACCTGTATCCCAAGCAG C SEQ ID NO: 7 CTCACCGCTGCTTGGGATA C SEQ ID NO: 8 TTATTTACTCACCGCTGCTT C SEQ ID NO: 9 AGAGTACACTGTATTTTTTG D SEQ ID NO: 10 GTCAATGCACAAGTTAGTGT D SEQ ID NO: 11 TTACGTTTACATTGCTCCAT D SEQ ID NO: 12 GTTATTTACTCACCGCTGCT D SEQ ID NO: 13 GAGCATTACAACAGCCATCG D SEQ ID NO: 14 CGATGGCTGTTGTAATGCTC E SEQ ID NO: 15 CTAACTTGTGCATTGACTAA E SEQ ID NO: 16 AATGCTCTGGTATCATGCAT E SEQ ID NO: 17 GAGGACTTATAATGTACTCA E SEQ ID NO: 18 TGTACTTACTTGGTTCTGTC E SEQ ID NO: 19 TTCCATCTTAAAATATATAC E SEQ ID NO: 20 AACCAGTATATATTTTAAGA E SEQ ID NO: 41 ATGTTAAGTTGTAGATAGTG B The KO efficiency is shown as A: >75%, B: 65-75%, C: 50-65%, D: 30-50%, E: <30%.

It should be understood that the implementation method section, rather than the content and abstract section, is intended to be used to interpret the scope of the application. The content and abstract section may set forth one or more but not all exemplary embodiments of the present disclosure contemplated by the inventor, and therefore are not intended to limit the scope of the present disclosure and the accompanying application in any way.

The foregoing description of specific embodiments will fully disclose the general nature of the present disclosure, so that others can easily modify and/or adapt various applications of such specific embodiments by applying the knowledge in this technology without departing from the general concept of the present disclosure, without undue experimentation. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the phrases or terms herein are for the purpose of explanation rather than limitation, so that skilled technicians should understand the terms or phrases of this specification according to the teachings and guidance.

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

The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) cited throughout this application are hereby expressly incorporated by reference as if fully written herein for any purpose, including the references cited therein. In the event of any inconsistency, the material disclosed in this text shall prevail.

Although various specific aspects have been shown and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the invention. After reading this description, many changes will become apparent to those skilled in the art.

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 wild-type ("WT") mesothelin-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) analyzed by flow cytometry. FIG1E 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 or MHC-I/MHC-II dKO meso iCAR-T cells, as indicated. FIG2 is a bar graph showing the cytotoxic effect of alloreactive ("allo")-T cells on (i) edited (WT); (ii) B2M/CIITA double KO; (iii) HLA-ABC/CIITA double KO; (iv) or HLA-ABC/CIITA double KO meso iCAR-T cells that further overexpress HLA-G and HLA-E. Cells were incubated for 24 hours at 2:1 and 1:1 effector:target (ET) ratios, as indicated. Figures 3A to 3D are line graphs showing NK cell-mediated killing of meso iCAR-T cells. NK cells were obtained from four donors (Donor 1: Figures 3A to 3D; Donor 2: Figures 3E to 3H; Donor 3: Figures 3I to 3L; and Donor 4: Figures 3M to 3P). meso iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) before co-culture with NK cells. meso iCAR-T cells were DKO (Fig. 3A, Fig. 3E, Fig. 3I, and Fig. 3M); DKO and PVR knockout (Fig. 3B, Fig. 3F, Fig. 3J, and 3N); DKO and CD58 knockout (Fig. 3C, Fig. 3G, Fig. 3K, and Fig. 3O); and DKO, PVR knockout, and CD58 knockout (Fig. 3D, Fig. 3H, Fig. 3L, and Fig. 3P). Cells were cultured for 24 hours at 5:1, 2:1, and 0:1 effector:target (E:T) ratios. The percentage of dead cells was assessed by FACS and analyzed using FlowJo software. "DKO" refers to B2M/CIITA double knockout. FIG4 is a line graph showing NK cell-mediated killing of meso iCAR-T cells. meso iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) and then co-cultured with NK cell lines at effector:target (E:T) ratios of 4:1, 2:1, and 1:1 for 24 hours. The percentage of dead meso iCAR-T 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 overexpression of FasDN. "+FasBB" refers to additional overexpression of Fas-41BB switch receptor. "+HSVTK" refers to additional overexpression of HSVTK. Figures 5A to 5C are line graphs showing NK cell-mediated killing of meso iCAR-T cells. NK cells were obtained from three donors (donor 1: Figure 5A; donor 2: Figure 5B; and donor 3: Figure 5C). meso iCAR-T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) and then co-cultured with NK cell lines at effector:target (E:T) ratios of 5:1 and 1.5:1 for 24 hours. The percentage of dead meso iCAR-T cells was evaluated by FACS and analyzed using FlowJo software. Figure 6 is a line graph showing tumor cell killing by 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, and 0.3:1 for 24 hours. The number of viable cells was assessed by the Cell Titer-Glo luminescent viability assay, which causes cell lysis and produces a luminescent signal proportional to the amount of ATP present. The luminescent signal was measured using a Pherastar plate reader. Figures 7A to 7C are line graphs showing NK cell-mediated killing of meso iCAR-T cells over time. FIG7A shows a normalized time course in which NSG-MHC I/II DKO animals were first given 1.5×10 ⁶ NK cells and then given WT (wild type, positive control); B2M and CIITA KO (untransduced or UTD); B2M/CIITA KO and overexpressing HLA-E and FasDN; HLA-ABC KO and overexpressing HLA-G; or HLA-ABC knockout and overexpressing HLA-E and FasDN iCAR T cells. iCAR T cells were administered at a dose of 1×10 ⁶ cells per animal. FIG7B to FIG7C show normalized time course data in which NSG-MHC I/II DKO animals were first given 2.0×10 ⁶ NK cells from two donors (FIG7B and FIG7C). One hour later, the animals were given 1×10 ⁶ cells per animal of WT (wild type); B2M/CIITA KO (untransduced or UTD); B2M/CIITA KO and overexpressing HLA-E and FasDN; B2M/CIITA/PVR KO and overexpressing HLA-E and FasDN; B2M/CIITA/CD58 KO and overexpressing HLA-E and FasDN; or B2M/CIITA/CD58/PVR KO and overexpressing HLA-E and FasDN iCART cells. Figures 8A to 8D are line graphs showing the persistence of iCART cells over time. Figure 8A shows NK cell-mediated killing of iCART cells further modified to overexpress FasBB and/or HSVTK killer switches. NSG-MHC I/II DKO animals were first given 2.0×10 ⁶ NK cells. One hour later, the animals were given iCARTs of WT (wild type); B2M/CIITA KO (untransduced or UTD; DKO-UTD); B2M/CIITA/PVR KO (TKO-UTD); B2M/CIITA/PVR KO (TKO) overexpressing HLA-E and FasDN; B2M/CIITA/CD58 KO (TKO) overexpressing HLA-E and HSVTK; B2M/CIITA/ PVR KO (TKO) overexpressing HLA-E, FasDN and HSVTK; B2M/CIITA/ PVR KO (TKO) overexpressing HLA-E, FasBB and HSVTK; each iCART was administered at a dose of 1×10 ⁶ cells per animal. Figure 8B shows data from the alloreactive T cell rejection assay. NSG-MHC I/II DKO mice were given 200 uL of freshly prepared alloreactive T cells intraperitoneally. One hour later, mice were administered 200 uL of iCAR T cells (WT; DKO-UTD; DKO and overexpressing HLA-E and FASDN; DKO and PVR KO and overexpressing HLA-E and FASDN; DKO and CD58 KO and overexpressing HLA-E and FASDN; or DKO and CD58 and PVR KO and overexpressing HLA-E and FASDN). Animals were imaged on IVIS Spectrum on days 1, 3, and 6 to obtain bioluminescent signals. The signal was then quantified as total flux (photons/second) within the region of interest (ROI). Figures 8C-8D show GSU tumor cell killing efficacy, where NSG-MHC I/II DKO mice were first given 1×10 ⁶ luciferized GSU cells, followed by 1×10 ⁶ cell doses per animal of WT; B2M/CIITA KO; B2M/CIITA KO and overexpressing HLA-E and FasDN; or B2M/CIITA/CD58 KO and overexpressing HLA-E and FasDN cells. Figures 9A-9B are schematic representations of the use of Fas receptors to affect apoptosis in modified immune cells. Figures 10A to 10F are graphical representations of cell proliferation of cells expressing mIL15/Ra (5BB) (Figure 10A), dnFAS (Figure 10B), FAS-4-1BB (Figure 10C), and FAS-OX40 (Figure 10D) after stimulation with FAS super ligands. Figure 10E is a bar graph showing the fold change in cell counts after addition of 0.1 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml super FAS ligands. Figure 10F shows the fold expansion of iCAR T cells and cells transduced with 5BB, dnFAS, FAS-4-1BB, and FAS-OX40 constructs during activation. Figures 11A to 11B are line graphs showing (i) untransduced cells prepared with boosted and non-boosted regimens (UTD, UTD-boosted); (ii) iCAR T cells expressing 5BB (5BB, 5BB-boosted) prepared with boosted and non-boosted regimens; (iii) dnFAS/5BB; and (iv) cell proliferation of FAS-OX40/5BB (Figure 11A) and cell lysis percentage of target cells (Figure 11B). Figures 12A to 12C are line graphs showing in vivo efficacy data of modified iCART cells in a mouse model. GSU-RFluc cells were intraperitoneally introduced into NSG-MHC I/II DKO mice at a dose of 1×10 ⁶ cells per animal. On day 4, iCAR-T cells were introduced at a dose of 1×10 ⁶ cells per animal, and cell lysis was analyzed based on mean fluorescence intensity. FIG. 12A shows the changes in cell lysis over time in the seven study 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). FIG. 12B to FIG. 12C show a comparison of the response of each animal to PBS and to 5BB ( FIG. 12B ) FAS-Ox40 ( FIG. 12C ). FIG. 13A to FIG. 13F are flow cytometric analysis diagrams of HLA-E expression in HLA-E knock-in iPSCs ( FIG. 13A ) and wild-type cells ( FIG. 13B ), FASDN expression in FASDN knock-in iPSCs ( FIG. 13C ) and wild-type cells ( FIG. 13D ), and tEGFR expression in tEGFR knock-in iPSCs ( FIG. 13E ) and wild-type cells ( FIG. 13F ).

TW202417617A_112130183_SEQL.xml

Claims (78)

A modified cell comprising: (i)      a chimeric antigen receptor (CAR) and/or an exogenous T cell receptor (eTCR); (ii)     endogenous MHC-I human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; (iii)    endogenous MHC-II human leukocyte antigen (HLA) expressed at reduced levels relative 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. The cell of claim 1, wherein the MHC-I human leukocyte antigens are HLA-A, HLA-B and HLA-C. The cell of claim 1, wherein the MHC-II human leukocyte antigens are HLA-DP, HLA-DQ and HLA-DR. The cell of claim 1, wherein the reduced expression of the MHC-I human leukocyte antigens is caused by a mutation or deletion of one or more endogenous genes encoding beta-2-microglobulin (B2M). The cell of claim 1, wherein the reduced expression of the endogenous MHC-II human leukocyte antigens is caused by a mutation or deletion of one or more endogenous genes encoding class II major histocompatibility complex transactivator protein (CIITA). The cell of claim 1, further comprising reduced expression of endogenous CD58 relative to wild-type cells of the same cell type. The cell of claim 6, wherein the reduced expression of CD58 is caused by mutation or deletion of one or more endogenous genes encoding CD58. The cell of any one of claims 1 to 7, comprising a nucleic acid encoding Fas-DN. A cell according to any one of claims 1 to 7, comprising a nucleic acid encoding Fas-CD27. A cell according to any one of claims 1 to 7, comprising a nucleic acid encoding Fas-BB. The cell of any one of claims 1 to 7, comprising a nucleic acid encoding Fas-OX40. A modified cell comprising: (i)      CAR or eTCR; (ii)     endogenous MHC-I human leukocyte antigen (HLA) with reduced expression relative to wild-type cells of the same cell type; (iii)    endogenous MHC-II human leukocyte antigen (HLA) with reduced expression relative to wild-type cells of the same cell type; and (iv)    Fas-DN. A cell comprising: (i)      CAR or eTCR; (ii)     endogenous MHC-I human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; (iii)    endogenous MHC-II human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; and (iv)    Fas-CD27. A cell comprising: (i)      CAR or eTCR; (ii)     endogenous MHC-I human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; (iii)    endogenous MHC-II human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; and (iv)    Fas-BB. A cell comprising: (i)      CAR or eTCR; (ii)     endogenous MHC-I human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; (iii)    endogenous MHC-II human leukocyte antigen (HLA) expressed at reduced levels relative to wild-type cells of the same cell type; and (iv)    Fas-OX40. The cell of any one of claims 12 to 15, further comprising reduced expression of endogenous CD58 relative to wild-type cells of the same cell type. The cell of claim 16, wherein the reduced expression of CD58 is caused by a mutation or deletion of one or more endogenous genes encoding CD58. A cell as in any one of claims 1 to 17, further comprising reduced expression of endogenous poliovirus receptor (PVR) relative to wild-type cells of the same cell type. The cell of claim 18, wherein the reduced expression of PVR is caused by a mutation or deletion of one or more endogenous genes encoding PVR. The cell of any one of claims 1 to 19, further comprising increased expression of HLA-E relative to wild-type cells of the same cell type. The cell of claim 20, wherein the increased expression of HLA-E is caused by: (i) transfection of a nucleic acid encoding an HLA-E polypeptide; (ii) modification of an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii). 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. A cell according to any one of claims 1 to 22, further comprising a human interleukin 15 (IL15) polypeptide. The cell of claim 23, wherein the human IL15 polypeptide is a membrane-bound IL15/IL15Rα fusion polypeptide (mIL15/Ra). A cell according to any one of claims 1 to 24, further comprising a human chemokine (C-C motif) ligand 19 (CCL19) polypeptide. The cell of 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. The cell of any one of claims 1 to 26, wherein the cell is (i) an immune cell differentiated from iPSC or (ii) a hematopoietic stem cell differentiated from iPSC. 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. A cell as in 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. The cell of claim 29, wherein the tumor antigen comprises CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-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, claudin, B7H3 or any combination thereof. A cell as in any one of claims 1 to 30, comprising a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to mesothelin. A method for engineering human cells, comprising: (i)      introducing a nucleic acid encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR) into the cell; (ii)     inactivating one or more endogenous genes encoding MHC-I human leukocyte antigen (HLA), wherein the inactivation results in reduced expression of MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (ii)     inactivating one or more endogenous genes encoding MHC-II human leukocyte antigen (HLA), wherein the inactivation results in reduced expression of MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iv)    introducing a nucleic acid encoding a 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 are introduced into the cell. The method of claim 32, further comprising: (v)     inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in a decrease in the expression of CD58 relative to wild-type cells of the same cell type. The method of claim 33, wherein the inactivation of one or more endogenous genes encoding CD58 comprises modifying the CD58 gene using a gene editing tool. The method of claim 33 or 34, wherein the gene editing tool comprises CRISPR/Cas9, CRISPR/Cas12, TALEN, zinc finger endonuclease or any combination thereof. The method of any one of claims 33 to 35, wherein inactivation of the one or more endogenous genes encoding CD58 comprises introducing CRISPR/Cas9 and a polynucleotide sequence encoding one or more guide sequences into the cell, wherein the one or more guide sequences hybridize with one or more target sequences within the CD58 gene. The method of claim 36, wherein the one or more guide sequences are hybridized with a nucleotide sequence in exon 3 of an endogenous CD58 gene. 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. The method of any one of claims 32 to 38, further comprising inactivating one or more endogenous genes encoding poliovirus receptor (PVR), wherein the inactivation results in reduced expression of PVR relative to wild-type cells of the same cell type. The method of any one of claims 32 to 39, further comprising (i) introducing a nucleic acid encoding an HLA-E polypeptide into the cells; (ii) modifying an endogenous gene encoding an HLA-E polypeptide, wherein the modification increases the expression of the endogenous HLA-E polypeptide; or (iii) both (i) and (ii). The method of claim 40, wherein the HLA-E polypeptide is a chimeric polypeptide comprising an HLA-E polypeptide linked to a B2M polypeptide. The method of any one of claims 32 to 41, further comprising transfecting the cell with a nucleic acid encoding an interleukin 15 (IL15) polypeptide. The method of claim 42, wherein the IL15 polypeptide is a membrane-bound IL15/IL15Rα fusion polypeptide (mIL15/Ra). The method of any one of claims 32 to 43, further comprising introducing a heterologous nucleic acid encoding a human interleukin (C-C motif) ligand 19 (CCL19) polypeptide into the cell. 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. The method of any one of claims 32 to 45, wherein the cell is an immune cell or a hematopoietic stem cell differentiated from iPSC. The method of any one of claims 32 to 46, wherein the cells comprise T cells, NK cells, NKT cells, or tumor infiltrating lymphocytes. 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. The method of claim 48, wherein the tumor antigen comprises CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-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, BCMA, GCC, ADGRE, claudin, or any combination thereof. A cell prepared according to the method of any one of claims 32 to 49. A cell according to any one of claims 1 to 31 and 50, which has increased in vivo persistence relative to a wild-type cell of the same cell type. A cell population comprising the cell of any one of claims 1 to 31, 50 and 51. A cell population, wherein at least 50% of the cells in the population comprise the cell of any one of claims 1 to 31, 50 and 51. A method for treating an individual in need thereof, comprising administering to the individual a cell according to any one of claims 1 to 31, 50 and 51, or a cell population according to claim 52 or 53. The method of claim 54, wherein the individual suffers from cancer. The method of claim 55, wherein the cancer comprises bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma 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, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem neurofibromas, pituitary adenomas, Kaposi's sarcoma (Kaposi's sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer including asbestos-induced cancer, or any combination thereof. A guide RNA capable of hybridizing with a human CD58 gene, comprising a nucleic acid sequence selected from SEQ ID NOs: 1-20 and 41. The guide RNA of claim 57, comprising the nucleic acid sequence shown in SEQ ID NO: 1. The guide RNA of claim 57, which consists of the nucleic acid sequence shown in SEQ ID NO: 1. The guide RNA of claim 57, comprising the nucleic acid sequence shown in SEQ ID NO: 2. The guide RNA of claim 57, which consists of the nucleic acid sequence shown in SEQ ID NO: 2. The guide RNA of claim 57, comprising the nucleic acid sequence shown in SEQ ID NO: 3. The guide RNA of claim 57, which consists of the nucleic acid sequence shown in SEQ ID NO: 3. The guide RNA of claim 57, comprising the nucleic acid sequence shown in SEQ ID NO: 4. The guide RNA of claim 57, which consists of the nucleic acid sequence shown in SEQ ID NO: 4. A method for inactivating a human CD58 gene in a cell, comprising contacting the cell with a guide RNA or a nucleic acid encoding the guide RNA as described in any one of claims 57 to 65 and a DNA endonuclease or a nucleic acid encoding the DNA endonuclease. The method of claim 59, wherein the DNA endonuclease comprises CRISPR/Cas9. A dominant negative Fas (Fas-DN) comprising the amino acid sequence shown in SEQ ID NO:27. A Fas-CD27 chimeric polypeptide (Fas-CD27) comprises the amino acid sequence shown in SEQ ID NO:22. A Fas-4-1BB chimeric polypeptide (Fas-4-1BB) comprises the amino acid sequence shown in SEQ ID NO: 23. A Fas-OX40 chimeric polypeptide (Fas-OX40) comprises the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26. The cell of claim 1, wherein the eTCR is a γ-δ TCR. The cell of claim 72, wherein the γ-δ TCR is Vγ9-Vδ2 TCR (g9d2TCR). A modified cell comprising: (i)      reduced expression of endogenous MHC-I human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; (ii)     reduced expression of endogenous MHC-II human leukocyte antigen (HLA) relative to wild-type cells of the same cell type; and (iii) increased expression of Fas (Fas-DN), HLA-E relative to wild-type cells of the same cell type, a nucleic acid encoding a suicide gene, or any combination thereof. A modified cell comprising: (i)      an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii)     an inactivated endogenous gene encoding 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. A modified cell comprising: (i)      an inactivated endogenous gene encoding MHC-I human leukocyte antigen (HLA); (ii)     an inactivated endogenous gene encoding 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. A method for engineering human cells comprising: (i)      inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of MHC-I human leukocyte antigens relative 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 reduced expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; and (iii)    transfecting the cell 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. A method for engineering human cells comprising: (i)      inactivating one or more endogenous genes encoding MHC-I human leukocyte antigens, wherein the inactivation results in reduced expression of MHC-I human leukocyte antigens relative 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 reduced expression of MHC-II human leukocyte antigens relative to wild-type cells of the same cell type; (iii)    inactivating one or more endogenous genes encoding CD58, wherein the inactivation results in reduced expression of CD58 relative to wild-type cells of the same cell type; and (iv)    using a gene encoding a dominant negative Fas The cell is transfected with nucleic acid encoding (Fas-DN), nucleic acid encoding exogenous HLA-E polypeptide, nucleic acid encoding suicide gene or any combination thereof.