WO2024153234A1

WO2024153234A1 - Engineered immune cells and uses thereof

Info

Publication number: WO2024153234A1
Application number: PCT/CN2024/073281
Authority: WO
Inventors: Ri Zhao; Lu MENG; Qiang Zou
Original assignee: Chengdu Ucello Biotechnology Co., Limited
Priority date: 2023-01-20
Filing date: 2024-01-19
Publication date: 2024-07-25

Abstract

Provided is an immune cell comprising one or more genetic alternations that reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK proteins, or a population of immune cells resulted from genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, wherein the immune cell or immune cell population exhibits (1) reduced MHC-I molecules on the cell surfaces of the immune cells, and (2) comparable or significantly improved tolerance to NK-cell mediated cytotoxicity than corresponding immune cells with β2M knockout. Methods to prepare the same and to use the same to treat a disease or a condition are also provided herein.

Description

ENGINEERED IMMUNE CELLS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of international patent application No. PCT/CN2023/073371, filed on January 20, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND

Universal cell therapy can be beneficial in a wide range of clinical contexts. However, universal cell is not without challenges.

A major obstacle to the clinical application of cell therapies is immune incompatibility. The host’s CD8+ cytotoxic T cells and CD4+ helper T cells can eliminate allogeneic cells by recognizing MHC-I molecules and MHC-П molecules on the cell membrane. Autologous cell transplantation can avoid the problem of immune rejection, but the personalized cell preparation method makes it an expensive therapeutic approach.

Since the first CAR-T cell therapy was approved for marketing in 2017, a total of six CAR-T cell therapies have been approved by regulatory authorities worldwide. However, all the currently approved CAR-T cells are autologous cells. Allogeneic universal CAR-T cell can be prepared using T cells from healthy donors, greatly shortening the time for patients to wait for treatment. In addition, the vitality and function of T cells obtained from healthy donors are usually better than patient-derived T cells. However, the problems faced during the development of allogeneic universal CAR-T cells include: (1) after the engineered CAR-T cells are injected into the patient's body, they can attack the patient's normal cells or tissues, resulting in graft-versus-host disease (GVHD) ; (2) the patient's own immune system can also reject allogeneic CAR-T cells, resulting in host-versus-graft reaction (HVGR) .

Although knocking out HLA molecules (e.g., MHC-I molecules) can ensure that injected cells (e.g., CAR-T cells) are not eliminated by the patient’s own T cells without using antibodies or other treatments, the cells with HLA molecules knocked out, particularly MHC-I molecules, can be recognized and rejected (or cleared) by the patient’s NK cells.

Therefore, there is still a need to improve allogeneic universal cells, e.g., to avoid the killing of injected cells (e.g., CAR-T cells) by the patient's own T lymphocytes, and to reduce the killing of injected cells (e.g., CAR-T cells) by NK cells, so as to reduce or avoid HVGR risk.

SUMMARY

Disclosed herein, in some aspects, is a population of immune cells resulted from genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, wherein at least 70%of immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene. In some cases, the at least 70%of the immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene. In some cases, the genetic engineering does not knock out β2M gene, does not knock down β2M gene, or both. In some cases, the population of immune cells does not have knockout of β2M gene. In some cases, at least 70%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of the one or more of RFX5, RFXAP, or RFXANK protein. In some cases, at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of one or more of RFX5, RFXAP, and RFXANK protein. In some cases, at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene. In some cases, the one or more genomic alterations comprise deletion, insertion, substitution, or any combination thereof. In some cases, the one or more genomic alterations result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene. In some cases, the population of immune cells comprises lymphocytes or myeloid cells. In some cases, the lymphocytes comprise T cells, B cells, tumor infiltrating lymphocytes, or natural killer cells. In some cases, the lymphocytes comprise CD8+ T cells or CD4+ T cells. In some cases, the T cells comprise a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) . In some cases, the lymphocytes comprise T cells, and the T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some cases, the population of immune cells is derived from peripheral blood, bone marrow, placenta, or umbilical cord. In some cases, the population of immune cells is derived from an autologous donor. In some cases, the population of immune cells is derived from an allogenic donor. In some cases, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not knock out a NK activating receptor ligand gene in the immune cell. In some cases, the population of immune cells does not have knockout of a NK activating receptor ligand gene, does not have knockdown of the NK activating receptor ligand gene, or both. In some cases, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cells. In some cases, the population of immune cells does not have a heterologous nucleic acid encoding a NK inhibitory molecule. In some cases, the percentage of the immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in the population is greater than the percentage of immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in a population of corresponding immune cells resulted from genetic engineering to knock out β2M gene. In some cases, the genetic engineering to knock out β2M gene does not knock out one or more of RFX5, RFXAP, or RFXANK gene. In some cases, the population of corresponding immune cells does not have knockout of one or more of RFX5, RFXAP, or RFXANK gene. In some cases, the population of immune cells has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to the population of corresponding immune cells. In some cases, the NK cell-mediated cellular cytotoxicity is measured by an assay in which the population of immune cells is contacted with NK cells. In some cases, the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord. In some cases, the population of immune cells has comparable tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells. In some cases, the population of immune cells has increased tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.

Disclosed herein, in some aspects, is a population of T cells comprising a CAR (CAR-T cells) , wherein: (i) the CAR-T cells have one or more genomic alternations that reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, (ii) at least 70%of the CAR-T cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene, (iii) the CAR-T cells do not have knockout of β2M gene, and (iv) the CAR-T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some cases, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε. In some cases, the CAR-T cells have a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.

Disclosed herein, in some aspects, is an immune cell that (i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein, and (ii) does not have knockout of β2M gene, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and/or HLA-C gene. In some cases, the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene. In some cases, the immune cell does not have knockdown of β2M gene. In some cases, the immune cell does not have knockout of β2M gene or knockdown of β2M gene. In some cases, the one or more genomic alterations comprise one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene. In some cases, the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene comprise deletion, insertion, substitution, or any combination thereof. In some cases, the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene. In some cases, the immune cell is a lymphocyte or a myeloid cell. In some cases, the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell. In some cases, the lymphocyte is a CD8+ T cell or a CD4+ T cell. In some cases, the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) . In some cases, the lymphocyte is a T cell, and the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some cases, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε. In some cases, the T cell has a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof. In some cases, the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord. In some cases, the immune cell is derived from an autologous donor. In some cases, the immune cell is derived from an allogenic donor. In some cases, the immune cell does not have knockout of a NK activating receptor ligand gene, does not have knockdown of the NK activating receptor ligand gene, or both. In some cases, the immune cell does not have a heterologous nucleic acid encoding a NK inhibitory molecule. In some cases, the immune cell has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to a corresponding immune cell that has knockout of β2M gene. In some cases, the corresponding immune cell does not have one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein. In some cases, the NK cell-mediated cellular cytotoxicity is measured by an assay in which the immune cell is contacted with NK cells. In some cases, the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord. In some cases, the immune cell has comparable tolerance to T cell-mediated cellular cytotoxicity, as compared to the corresponding immune cell.

Disclosed herein, in some aspects, is a T cell comprising a CAR (CAR-T cell) , wherein the CAR-T cell (i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein, (ii) does not have knockout of β2M gene, and (iii) has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some cases, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.

Disclosed herein, in some aspects, is a method of reducing expression of HLA-A, HLA-B, or HLA-C gene while maintaining tolerance of an immune cell to host NK cells, comprising genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein in the immune cell. In some cases, the method does not comprise knocking out β2M gene in the immune cell, does not comprise knocking down of the β2M gene in the immune cell, or both. In some cases, the method does not comprise knocking out a NK activating receptor ligand gene in the immune cell, does not comprise knocking down the NK activating receptor ligand gene in the immune cell, or both. In some cases, the method does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cell. In some cases, the immune cell is a lymphocyte or a myeloid cell. In some cases, the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell. In some cases, the lymphocyte is a CD8+ T cell or a CD4+ T cell. In some cases, the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) . In some cases, the lymphocyte is a T cell, and the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some cases, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε. In some cases, the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord. In some cases, the immune cells are derived from an autologous donor. In some cases, the immune cells are derived from an allogenic donor.

Disclosed herein, in some aspects, is a method comprising administering the population of immune cells described herein, the population of CAR-T cells described herein, the immune cell described herein, or the CAR-T cell described herein, to a subject in need thereof. In some cases, the disease or condition is cancer, optionally acute lymphoblastic leukemia (ALL) .

Disclosed herein, in some aspects, is a pharmaceutical composition comprising the population of immune cells described herein, the population of CAR-T cells described herein, the immune cell described herein, or the CAR-T cell described herein, and a pharmaceutically acceptable excipient or carrier.

Disclosed herein, in some aspects, is use of the population of immune cells described herein, the population of CAR-T cells described herein, the immune cell described herein, or the CAR-T cell described herein in an adoptive cell therapy.

Disclosed herein, in some aspects, is a guide RNA (gRNA) or a polynucleotide encoding the guide RNA, the guide RNA comprising a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-16.

Disclosed herein, in some aspects, is a ribonucleoprotein (RNP) complex comprising the gRNA described herein and a Cas protein.

Disclosed herein, in some aspects, is a composition comprising the gRNA described herein. In some cases, the composition comprises an immune cell that contains the gRNA. In some cases, the immune cell further comprises an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) .

Disclosed herein, in some aspects, is a method of engineering a cell, comprising introducing the gRNA described herein into the cell, or contacting the cell with the RNP complex described herein. In some cases, the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-8; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-8, thereby resulting in genomic alteration of RFX5 gene in the cell. In some cases, the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 9-12; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 9-12, thereby resulting in genomic alteration of RFXANK gene in the cell. In some cases, the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 13-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 13-16, thereby resulting in genomic alteration of RFXAP gene in the cell. In some cases, the method comprises electroporating the cell with reagents comprising the gRNA or the RNP complex. In some cases, the method further comprises contacting the cell with a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) . In some cases, the nucleic acid molecule is a vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some cases, the cell is an immune cell, optionally a CD4+ T cell or a CD8+ T cell. In some cases, the cell is isolated from cord blood of a human or is a progeny of a cell isolated from cord blood of a human.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B show the survival and proliferation of RFX5-KO umbilical cord blood CD4-positive (CD4+) T cells, as compared to corresponding β2M-KO umbilical cord blood CD4+ T cells, in the presence of autologous umbilical cord blood NK cells. The following abbreviations are used in FIGs. 1A-6B: “CD4-O-ABC+” stands for control HLA-ABC positive CD4+ T cells; “CD4-β2M-ABC+” stands for HLA-ABC positive CD4+T cells after knocking out β2M using β2M gRNA; “CD4-β2M-ABC-” stands for HLA-ABC negative CD4+T cells after knocking out β2M using β2M gRNA; “CD4-RFX5-ABC+” stands for HLA-ABC positive CD4+T cells after knocking out RFX5 using 4R-GH gRNA; “CD4-RFX5-ABC-” stands for HLA-ABC negative CD4+T cells after knocking out RFX5 using 4R-GH gRNA; “CD8-O-ABC+” stands for control HLA-ABC positive CD8+ T cells; “CD8-β2M-ABC+” stands for HLA-ABC positive CD8+T cells after knocking out β2M using β2M gRNA; “CD8-β2M-ABC-” stands for HLA-ABC negative CD8+T cells after knocking out β2M using β2M gRNA; “CD8-RFX5-ABC+” stands for HLA-ABC positive CD8+T cells after knocking out RFX5 using 4R-GH gRNA; “CD8-RFX5-ABC-” stands for HLA-ABC negative CD8+T cells after knocking out RFX5 using 4R-GH gRNA. FIG. 1A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in RFX5-KO umbilical cord blood CD4+ T cells, as compared to that in β2M-KO umbilical cord blood CD4+ T cells, in the presence of autologous umbilical cord blood NK cells at an effector-target ratio (E: T) of 1: 1, at days 0, 3, and 6. FIG. 1B shows the fold expansion of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in RFX5-KO umbilical cord blood CD4+ T cells, as compared to that in β2M-KO umbilical cord blood CD4+ T cells and the fold expansion of control HLA-ABC positive CD4+T cells, in the presence of autologous umbilical cord blood NK cells at an E: T ratio of 1: 1 at days 0, 3, and 6.

FIGs. 2A-2B show the survival and proliferation of RFX5-KO umbilical cord blood CD8+ T cells, as compared to corresponding β2M-KO umbilical cord blood CD8+ T cells, in the presence of autologous umbilical cord blood NK cells. FIG. 2A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in RFX5-KO umbilical cord blood CD8+ T cells, as compared to that in β2M-KO umbilical cord blood CD8+ T cells, in the presence of autologous umbilical cord blood NK cells at an effector-target ratio (E: T) of 1: 1, at days 0, 3, and 6. FIG. 2B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in RFX5-KO umbilical cord blood CD8+ T cells, as compared to that in β2M-KO umbilical cord blood CD8+ T cells and the fold expansion of control HLA-ABC positive CD8+ T cells, in the presence of autologous umbilical cord blood NK cells at an E: T ratio of 1: 1 at days 0, 3, and 6.

FIGs. 3A-3B show the survival and proliferation of RFX5-KO umbilical cord blood CD4+ T cells, as compared to corresponding β2M-KO umbilical cord blood CD4+ T cells, in the presence of allogeneic umbilical cord blood NK cells. FIG. 3A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in RFX5-KO umbilical cord blood CD4+ T cells, as compared to that in β2M-KO umbilical cord blood CD4+ T cells, in the presence of allogeneic umbilical cord blood NK cells at an effector-target ratio (E: T) of 1: 1, at days 0, 3, and 6. FIG. 3B shows the fold expansion of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in RFX5-KO umbilical cord blood CD4+ T cells, as compared to that in β2M-KO umbilical cord blood CD4+ T cells and the fold expansion of control HLA-ABC positive CD4+ T cells, in the presence of allogeneic umbilical cord blood NK cells at an E: T ratio of 1: 1 at days 0, 3, and 6.

FIGs. 4A-4B show the survival and proliferation of RFX5-KO peripheral blood CD4+ T cells, as compared to corresponding β2M-KO peripheral blood CD4+ T cells, in the presence of autologous peripheral blood NK cells. FIG. 4A shows the percentage of cells that are CD4+ ABC-and CD4+ABC+, respectively, in RFX5-KO peripheral blood CD4+ T cells, as compared to that in β2M-KO peripheral blood CD4+ T cells, in the presence of autologous peripheral blood NK cells at an effector-target ratio (E: T) of 1: 1, at days 0, 3, and 6. FIG. 4B shows the fold expansion of cells that are CD4+ABC-and CD4+ ABC+, respectively, in RFX5-KO peripheral blood CD4+ T cells, as compared to that in β2M-KO peripheral blood CD4+ T cells and the fold expansion of control HLA-ABC positive CD4+T cells, in the presence of autologous peripheral blood NK cells at an E: T ratio of 1: 1 at days 0, 3, and 6.

FIGs. 5A-5B show the survival and proliferation of RFX5-KO peripheral blood CD8+ T cells, as compared to corresponding β2M-KO peripheral blood CD8+ T cells, in the presence of autologous peripheral blood NK cells. FIG. 5A shows the percentage of cells that are CD8+ ABC-and CD8+ABC+, respectively, in RFX5-KO peripheral blood CD8+ T cells, as compared to that in β2M-KO peripheral blood CD8+ T cells, in the presence of autologous peripheral blood NK cells at an effector-target ratio (E: T) of 1: 1, at days 0, 3, and 6. FIG. 5B shows the fold expansion of cells that are CD8+ABC-and CD8+ ABC+, respectively, in RFX5-KO peripheral blood CD8+ T cells, as compared to that in β2M-KO peripheral blood CD8+ T cells and the fold expansion of control HLA-ABC positive CD8+T cells, in the presence of autologous peripheral blood NK cells at an E: T ratio of 1: 1 at days 0, 3, and 6.

FIGs. 6A-6B show the survival and proliferation of RFX5-KO umbilical cord blood CD8+ T cells, as compared to corresponding β2M-KO umbilical cord blood CD8+ T cells, in the presence of allogeneic peripheral blood NK cells. FIG. 6A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in RFX5-KO umbilical cord blood CD8+ T cells, as compared to that in β2M-KO umbilical cord blood CD8+ T cells, in the presence of allogeneic peripheral blood NK cells at an effector-target ratio (E: T) of 1: 1, at days 0, 3, and 6. FIG. 6B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in RFX5-KO umbilical cord blood CD8+ T cells, as compared to that in β2M-KO umbilical cord blood CD8+ T cells and the fold expansion of control HLA-ABC positive CD8+ T cells, in the presence of allogeneic peripheral blood NK cells at an E: T ratio of 1: 1 at days 0, 3, and 6.

FIGs. 7A-7B show the survival and proliferation of RFX5-KO CD4+ T cells, as compared to corresponding RFXANK-KO, RFXAP-KO, CIITA-KO, and β2M-KO CD4+ T cells, in the presence of NK cells. The following abbreviations are used in FIGs. 7A-8B: “4-O” stands for control CD4+ T cells; “4-β2M” stands for HLA-ABC positive or negative CD4+T cells after knocking out β2M using β2M gRNA; “4-4R-GH” stands for HLA-ABC positive or negative CD4+T cells after knocking out RFX5 using the 4R-GH gRNA; “4-5R-CD” stands for HLA-ABC positive or negative CD4+T cells after knocking out RFXANK using 5R-CD gRNA; “4-6R-CD” stands for HLA-ABC positive or negative CD4+T cells after knocking out RFXAP using 6R-CD gRNA; “4-3C-AB” stands for HLA-ABC positive or negative CD4+T cells after knocking out CIITA using 3C-AB gRNA; “8-O” stands for control CD8+T cells; “8-M” stands for HLA-ABC positive or negative CD8+T cells after knocking out β2M using β2M gRNA; “8-4R-GH” stands for HLA-ABC positive or negative CD8+T cells after knocking out RFX5 using 4R-GH gRNA; “8-5R-CD” stands for HLA-ABC positive or negative CD8+T cells after knocking out RFXANK using 5R-CD gRNA; “8-6R-CD” stands for HLA-ABC positive or negative CD8+T cells after knocking out RFXAP using 6R-CD gRNA; “8-3C-AB” stands for HLA-ABC positive or negative CD8+T cells after knocking out CIITA using 3C-AB gRNA. FIG. 7A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of a respective gene using gRNAs targeting β2M (β2M-sgRNA) , CIITA (3C-AB) , RFX5 (4R-GH) , RFXANK (5R-CD) , and RFXAP (6R-CD) before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 7B shows the fold expansion of cells that are CD4+ ABC-and CD4+ABC+, respectively, in CD4+ T cells with a knockout of a respective gene using gRNAs targeting β2M (β2M-sgRNA) , CIITA (3C-AB) , RFX5 (4R-GH) , RFXANK (5R-CD) , and RFXAP (6R-CD) , as compared to control CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 8A-8B show the survival and proliferation of RFX5-KO CD8+ T cells, as compared to corresponding RFXANK-KO, RFXAP-KO, CIITA-KO, and β2M-KO CD8+ T cells, in the presence of NK cells. FIG. 8A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of a respective gene using gRNAs targeting β2M (β2M-sgRNA) , CIITA (3C-AB) , RFX5 (4R-GH) , RFXANK (5R-CD) , and RFXAP (6R-CD) before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 8B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of a respective gene using gRNAs targeting β2M (β2M-sgRNA) , CIITA (3C-AB) , RFX5 (4R-GH) , RFXANK (5R-CD) , and RFXAP (6R-CD) , as compared to control CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 9A-9B show the survival and proliferation of RFX5-KO CD4+ T cells, as compared to corresponding β2M-KO CD4+ T cells, in the presence of NK cells, wherein RFX5-KO was performed using different gRNAs. The following abbreviations are used in FIGs. 9A-14B: “4-O” stands for control CD4+ T cells; “4-β2M” stands for HLA-ABC positive or negative CD4+T cells after knocking out β2M using β2M-sgRNA; “4-4R-AB, ” “4-4R-CD, ” “4-4R-EF, ” “4-4R-GH, ” and “4-4R-IJ” stands for HLA- ABC positive or negative CD4+T cells after knocking out RFX5 at different sites using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively; “4-5R-AB, ” “4-5R-CD, ” “4-5R-EF, ” and “4-5R-GH” stands for HLA-ABC positive or negative CD4+T cells after knocking out RFXANK at different sites using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively; “4-6R-AB, ” “4-6R-CD, ” “4-6R-EF, ” “4-6R-GH” stands for HLA-ABC positive or negative CD4+T cells after knocking out RFXAP at different sites using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, respectively; “8-O” stands for control CD8+ T cells; “8-M” stands for HLA-ABC positive or negative CD8+T cells; “8-4R-AB, ” “8-4R-CD, ” “8-4R-EF, ” “8-4R-GH, ” and “8-4R-IJ” stands for HLA-ABC positive or negative CD8+T cells after knocking out RFX5 at different sites using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively; “8-5R-AB, ” “8-5R-CD, ” “8-5R-EF, ” and “8-5R-GH” stands for HLA-ABC positive or negative CD8+T cells after knocking out RFXANK at different sites using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively; “8-6R-AB, ” “8-6R-CD, ” “8-6R-EF, ” and “8-6R-GH” stands for HLA-ABC positive or negative CD8+T cells after knocking out RFXAP at different sites using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, respectively. FIG. 9A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFX5 at different sites using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively, as compared to that in β2M-KO CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 9B shows the fold expansion of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFX5 at different sites using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively, as compared to that in β2M-KO CD4+ T cells and control CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 10A-10B show the survival and proliferation of RFX5-KO CD8+ T cells, as compared to corresponding β2M-KO CD8+ T cells, in the presence of NK cells, wherein RFX5-KO was performed using different gRNAs. FIG. 10A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFX5 at different sites using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively, as compared to that in β2M-KO CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 10B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFX5 at different sites using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively, as compared to that in β2M-KO CD8+ T cells and control CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 11A-11B show the survival and proliferation of RFXANK-KO CD4+ T cells, as compared to corresponding β2M-KO CD4+ T cells, in the presence of NK cells, wherein RFXANK-KO was performed using different gRNAs. FIG. 11A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFXANK at different sites using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively, as compared to that in β2M-KO CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 11B shows the fold expansion of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFXANK at different sites using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively, as compared to that in β2M-KO CD4+ T cells and control CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 12A-12B show the survival and proliferation of RFXANK-KO CD8+ T cells, as compared to corresponding β2M-KO CD8+ T cells, in the presence of NK cells, wherein RFXANK-KO was performed using different gRNAs. FIG. 12A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFXANK at different sites using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively, as compared to that in β2M-KO CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 12B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFXANK at different sites using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively, as compared to that in β2M-KO CD8+ T cells and control CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 13A-13B show the survival and proliferation of RFXAP-KO CD4+ T cells, as compared to corresponding β2M-KO CD4+ T cells, in the presence of NK cells, wherein RFXAP-KO was performed using different gRNAs. FIG. 13A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFXAP at different sites using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, respectively, as compared to that in β2M-KO CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 13B shows the fold expansion of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFXAP at different sites using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, respectively, as compared to that in β2M-KO CD4+ T cells and control CD4+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 14A-14B show the survival and proliferation of RFXAP-KO CD8+ T cells, as compared to corresponding β2M-KO CD8+ T cells, in the presence of NK cells, wherein RFXAP-KO was performed using different gRNAs. FIG. 14A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFXAP at different sites using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, respectively, as compared to that in β2M-KO CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6. FIG. 14B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFXAP at different sites using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, respectively, as compared to that in β2M-KO CD8+ T cells and control CD8+ T cells, before (day 0) and after mixing with NK cells at an effector-target ratio (E: T) of 1: 1 at days 3 and 6.

FIGs. 15A-15B show the survival and proliferation of RFX5-GH-KO CD4+ CAR-T cells, as compared to corresponding β2M-KO CD4+ CAR-T cells, in the presence of allogenic T cells. FIG. 15A shows the percentage of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFX5 using gRNA 4R-GH as compared to that in β2M-KO CD4+ T cells, before (day 0) and after mixing with allogenic T cells at an effector-target ratio (E: T) of 4: 1, 2: 1 and 1: 1, respectively, at days 3 and 6. FIG. 15B shows the fold expansion of cells that are CD4+ ABC-and CD4+ ABC+, respectively, in CD4+ T cells with a knockout of RFX5 using gRNA 4R-GH, as compared to that in β2M-KO CD4+ T cells, TCR-KO CD4+ T cells, and control CD4+ T cells, before (day 0) and after mixing with allogenic T cells at an effector-target ratio (E: T) of 4: 1, 2: 1 and 1: 1, respectively, at days 3 and 6.

FIGs. 16A-16B show the survival and proliferation of RFX5-GH-KO CD8+ CAR-T cells, as compared to corresponding β2M-KO CD8+ CAR-T cells, in the presence of allogenic T cells. FIG. 16A shows the percentage of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFX5 using gRNA 4R-GH as compared to that in β2M-KO CD8+ T cells, before (day 0) and after mixing with allogenic T cells at an effector-target ratio (E: T) of 4: 1, 2: 1 and 1: 1, respectively, at days 3 and 6. FIG. 16B shows the fold expansion of cells that are CD8+ ABC-and CD8+ ABC+, respectively, in CD8+ T cells with a knockout of RFX5 using gRNA 4R-GH, as compared to that in β2M-KO CD8+ T cells, TCR-KO CD8+ T cells, and control CD8+ T cells, before (day 0) and after mixing with allogenic T cells at an effector-target ratio (E: T) of 4: 1, 2: 1 and 1: 1, respectively, at days 3 and 6.

FIG. 17 shows the efficiency of CAR19 lentivirus transduction in CD4+ or CD8+ CAR-T cells according to some embodiments of the present disclosure. “NoEP” stands for T cells without gene editing; “EP” stands for T cells with gene editing (RFX5-GH-KO) .

FIG. 18 shows killing of Raji cells measured via %cell lysis, by CD4+ CAR-T cells without gene editing, CD4+ and RFX-GH-KO CAR-T cells, CD8+ CAR-T cells without gene editing, and CD8+and RFX-GH-KO CAR-T cells, respectively, after being mixed at an E: T ratio of 1: 1, 1: 3 and 1: 9 respectively, according to some embodiments of the present disclosure.

FIG. 19 shows expression of CD107a in CD4+ CAR-T cells without gene editing, CD4+ and RFX-GH-KO CAR-T cells, CD8+ CAR-T cells without gene editing, and CD8+ and RFX-GH-KO CAR-T cells, respectively, after being mixed with Raji cells at an E: T ratio of 1: 1, 1: 3 and 1: 9 respectively, according to some embodiments of the present disclosure.

FIGs. 20A-20E show target gene (HLA-ABC) knockout efficiency using different gRNAs. FIG. 20A shows HLA-ABC knockout efficiency in CD4+ T cells using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively, each targeting RFX5. FIG. 20B shows HLA-ABC knockout efficiency in CD8+ T cells using gRNAs 4R-AB, 4R-CD, 4R-EF, 4R-GH, and 4R-IJ, respectively, targeting RFX5. FIG. 20C shows HLA-ABC knockout efficiency in CD4+ T cells and CD8+ T cells, respectively, using gRNAs 5R-AB, 5R-CD, 5R-EF, and 5R-GH, respectively, each targeting RFXANK. FIG. 20D shows HLA-ABC knockout efficiency in CD4+ T cells and CD8+ T cells, respectively, using gRNAs 6R-AB, 6R-CD, 6R-EF, 6R-GH, each targeting RFXAP. FIG. 20E shows a comparison of the HLA-ABC knockout efficiency of gRNAs targeting RFX5 (RFX5-GH-KO) , RFXANK (RFXANK-CD-KO) , RFXAP (RFXAP-CD-KO) , each of which is the best in knocking out HLA-ABC among the group targeting a given gene (i.e., RFX5, RFXANK, or RFXAP) .

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present disclosure.

The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y. ) ; MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press) ; MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique , 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986) ) ; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory) ; Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London) ; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, all of which are herein incorporated by reference in their entirety for all purposes.

The present disclosure provides, in some aspects, compositions, methods, and kits related to allogeneic adoptive cell therapy using cells with at least one genomic alterations that reduce or eliminate expression or function of a protein. In some cases, the cells are immune cells, for example, engineered immune cells. In some cases, the cells are engineered T cells. In some cases, the cells are CAR-T cells (e.g., CAR19 as described herein) . In some cases, the genomic alterations are carried out via CRISPR/Cas9 multiplex gene editing technology. In some cases, the cells comprise one or more genomic alterations targeting one or more genes. In some cases, the one or more genomic alterations comprise reduced or eliminated expression and/or function of one or more of RFX5, RFXAP, and RFXANK proteins. In some cases, the one or more genomic alterations comprise reduced or eliminated expression and/or function of one or more of RFX5, RFXAP, and RFXANK proteins. In some case, the one or more genomic alterations comprise reduced or eliminated expression of a gene encoding one or more of RFX5, RFXAP, and RFXANK proteins. In some case, the one or more genomic alterations comprise reduced or eliminated expression of RFX5 protein. The present disclosure demonstrates that genetic alternation (e.g., RFX5, RFXAP, and RFXANK gene knockout) that reduces or eliminates expression and/or function of one or more of RFX5, RFXAP, and RFXANK proteins in immune cells results in significant reduction not only in MHC-II molecules expression but also MHC-I molecules on the cell surfaces of the immune cells.

The present disclosure demonstrates that mere genetic alternation that reduces or eliminates expression and/or function of one or more of RFX5, RFXAP, and RFXANK proteins in immune cells results in significant reduction not only in MHC-II molecules expression but also MHC-I molecules on the cell surfaces of the immune cells. In particular, the genetic alternation achieves comparable or better reduction of MHC-I molecules in the immune cells, as compared to β2M-knockout. Unexpectedly, the resulting immune cells exhibits significantly improved tolerance to NK-cell mediated cytotoxicity as compared to corresponding immune cells with β2M knockout.

Definitions

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

In the present disclosure, wherever aspects are described herein with the language "comprising, " otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided. All definitions herein described whether specifically mentioned or not, should be construed to refer to definitions as used throughout the specification and attached claims.

Throughout the specification and attached claims, the singular form “a” , “an” , and “the” include plural references unless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain” ) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule. In some cases, the set of polypeptides are contiguous with each other, e.g., are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some embodiments, the set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In some cases, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some cases, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some cases, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137) , CD27 and/or CD28. In some cases, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain.

In the present disclosure, an “antibody” refers to an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The term as used herein, can include an immunoglobulin molecule that specifically binds to an antigen and comprises an FcR binding site which may or may not be functional. As used in the disclosure, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F (ab') 2, diabodies) Fv fragments and single chain (ScFv) mutants that contain an antigen recognition site or antigen binding site and have ability to bind to an antigen. Antigen-binding antibody or immunoglobulin fragments are well known in the art; such fragment can have a functional or non-functional Fc receptor binding site. Further as used herein, the term is not limited only to intact polyclonal or monoclonal antibodies, multispecific antibodies such as bispecific, or polyspecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted antibodies, human antibodies, and any other modified immunoglobulin molecule comprising an antigen binding site so long as the antibodies exhibit the desired biological activity.

A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD) ) ; and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948) ) . As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.

A “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

The term “cell population” or "population of cells, " as used herein interchangeably, refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1×10⁶ cells, at least about 1×10⁷ cells, at least about 1×10⁸ cells, at least about 1×10⁹ cells, at least about 1×10¹⁰ cells, at least about 1×10¹¹ cells, at least about 1×10¹² cells, or more cells expressing similar or different phenotypes.

The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term “effective amount” also applies to a dose that will provide an image for detection by an appropriate imaging method. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried. An effective amount of an active agent can be administered in a single dose or in multiple doses.

A “fragment” when applied to a protein or polypeptide, is a truncated form of a native biologically active protein or polypeptide that may or may not retain at least a portion of the therapeutic and/or biologic activity of the native biologically active protein or polypeptide. In some embodiments of the present disclosure, a “fragment” of a native biologically active protein or polypeptide retains at least a portion of the therapeutic and/or biologic activity of the native biologically active protein or polypeptide disclosed herein.

The term “gene editing system” or “genome editing system” refers to a system of one or more molecules comprising at least a nuclease (or nuclease domain) and a programmable nucleotide binding domain, which are necessary and sufficient to direct and effect modification (e.g., single or double-strand break) of nucleic acid at a target sequence by the nuclease (or nuclease domain) . In some embodiments, the gene editing system is a CRISPR system. In some embodiments, the gene editing system is a zinc finger nuclease (ZFN) system. In some embodiments, the gene editing system is a TALEN system. In some embodiments, the gene editing system is a meganuclease system. Those methods are well-known to a skilled in the art.

The term “CRISPR system, ” “Cas system, ” or “CRISPR/Cas system” refers to a set of molecules comprising an RNA-guided nuclease or other effector molecule and a guide RNA molecule that together are necessary and sufficient to direct and effect modification of nucleic acid at a target sequence by the RNA-guided nuclease or other effector molecule. In one embodiment, a CRISPR system comprises a guide RNA molecule and a Cas protein, e.g., a Cas9 protein. In one example, the guide RNA molecule and Cas molecule can be complexed, to form a ribonuclear protein (RNP) complex. The gRNA in the RNP guides and triggers Cas9 to cleave the double-stranded DNA target, activating non-homologous end joining (NHEJ) or creating a site for possible insertion of exogenous donor DNA through homology-directed repair (HDR) mechanisms.

The terms “guide RNA, ” “guide RNA molecule, ” “gRNA molecule” and “gRNA” are used interchangeably, and refer to a set of synthetic or recombinant nucleic acid molecules that promote the specific directing of an RNA-guided nuclease or other effector molecule (typically in complex with the gRNA molecule) to a target sequence. A gRNA molecule may have a number of domains, as described more fully below. In some embodiments, a gRNA molecule comprises a targeting domain and interacts with a Cas molecule, such as Cas9 or with another RNA-guided endonuclease such as Cpf1. In some embodiments, a gRNA molecule comprises a crRNA domain (comprising a targeting domain) and a tracr, e.g., for interacting with a Cas molecule such as Cas9. In some embodiments, directing of nuclease binding is accomplished through hybridization of a portion of the gRNA to DNA (e.g., through the gRNA targeting domain) , and by binding of a portion of the gRNA molecule to the RNA-guided nuclease or other effector molecule (e.g., through at least the gRNA tracr) . In some embodiments, the crRNA and the tracr are provided on a single contiguous polynucleotide molecule, referred to herein as a “single guide RNA, ” “sgRNA, ” or “single-molecule DNA-targeting RNA” and the like. In other embodiments, the crRNA and tracr are provided on separate polynucleotide molecules, which are themselves capable of association, usually through hybridization, referred to herein as a “dual guide RNA, ” “dgRNA, ” or “double-molecule DNA-targeting RNA” and the like. In some embodiments of dgRNAs, the crRNA and tracr are linked by a nonnucleotide chemical linker.

An “individual” or a "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

“Nucleic acid” or “Polynucleotide, ” as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps” , substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc. ) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc. ) , those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc. ) , those with intercalators (e.g., acridine, psoralen, etc. ) , those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc. ) , those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc. ) , as well as unmodified forms of the polynucleotide (s) . Further, any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-O-methyl-, 2’-O-allyl, 2’-fluoro-or 2’-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P (O) S ( “thioate” ) , P (S) S ( “dithioate” ) , (O) NR₂ ( “amidate” ) , P (O) R, P (O) OR’, CO or CH₂ ( “formacetal” ) , in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000) .

The terms “polypeptide” , “oligopeptide” , “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it may comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc. ) , as well as other modifications known in the art. It is understood that, when the polypeptides as described herein are based upon an antibody, the polypeptides can occur as single chains or associated chains.

A “variant” when applied to a protein is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99%amino acid sequence identity compared with the reference biologically active protein or any ranges in between the at least 70%and 99%. Variants can include, for example, an alteration, substitution, deletion, addition, or chemical modification of one or more amino acids, one or more unnatural amino acids, or any combination thereof of a parent peptide, , and can still retain the ability to specifically bind to the respective receptor, activate the downstream targets, and/or induce one or more of the differentiation, proliferation (or death) and activity of cells, e.g., T cells and NK cells, to a similar extent, the same extent, or to a higher extent, as the parent peptide.

In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide

In the context of polynucleotides, a “linear sequence” or a “sequence” is an order of nucleotides in a polynucleotide in a 5' terminus to 3' terminus direction in which nucleotides that neighbor each other in the sequence are contiguous in the primary structure of the polynucleotide.

An antibody or a CAR that "specifically binds" to an epitope is a term well understood in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell, protein or substance than it does with alternative cells, proteins or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to CD19 is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. As a further example, an antibody (or another moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

As used herein, "vector" means a construct, which is capable of delivering, and preferably expressing, one or more gene (s) or sequence (s) of interest in a host cell, such as an engineered cell or population of cells as described herein. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

The sequence identity with respect to the gRNA, CAR19, or any other amino acid or polynucleotide sequencings or nucleic acid sequences identified herein, is defined as the percentage of amino acid residues (or nucleotides) in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequencing or a portion thereof (or the nucleotides of a second, reference nucleic acid sequence or a portion thereof) , after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequencing or nucleic acid sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequencing, or larger, defined nucleic acid sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues or base pairs or nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. In some embodiments, percent identity is determined with respect to the full length of a noted reference sequence, such as a sequence provided herein. For example, sequence comparison between two amino acid sequences (or a shorter length thereof) of the present disclosure may be carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI) . The percentage amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has a certain %amino acid sequence identity to a given amino acid sequence B) is calculated by the formula as follows:

where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program’s alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter. Two polynucleotide or polypeptide sequencings are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.

Reducing Host-Versus-Graft Reactions (HvGR)

The present disclosure provides, in some aspects, compositions, methods, and kits related to allogeneic adoptive cell therapy with reduced or eliminated HvGR. HvGR can be caused by recognition by host immune system of MHC-class I and MHC-class II molecules expressed on the membrane of the allogenic cells (e.g., T cells expressing a CAR, not limited to CAR19 described herein, that is intended to be administered to a subject that is not the donor) , which can in turn result in killing of the allogenic cells by the immune cells of the subject. HLA expression on allogeneic T cells can be recognized by the host immune system as non-self, leading to rapid clearance by the immune cells of the subject through a rejection reaction. Knocking out RFX5, RFXAP, and/or RFXANK gene can reduce the expression of both HLA Type I and Type II molecules on the allogeneic cells.

The present disclosure provides an immune cell (e.g., an engineered immune cell or a CAR-T cell) or a population of immune cells (e.g., CAR-T cells) comprising one or more genomic alterations that result in reduced or eliminated HvGR. The immune cell or the population of immune cells can survive longer after administered to a subject receiving the allogeneic adoptive cell therapy as compared to allogeneic cells without such one or more genomic alterations.

MHC-I molecules or/and MHC-П molecules

HLA genes can be divided into MHC-I class and MHC-II class. MHC-I genes (including HLA-A, HLA-B, and HLA-C) are expressed in almost all tissue cell types, presenting "non-self" antigen peptides to allogeneic CD8+ T cells, thereby promoting their activation into cytotoxic CD8+ T cells. Transplanted or implanted cells expressing "non-self" MHC class I molecules will elicit a cellular immune response against these cells, ultimately leading to the killing of the implanted cells through activated cytolytic CD8+ T cells. MHC-I proteins are closely associated with β2 microglobulin (β2M) in the endoplasmic reticulum, which can be critical for the formation of functional MHC class I molecules on the cell surface.

In contrast to the universal cellular expression of MHC-I genes, the expression of MHC-П genes is restricted to antigen-presenting cells, such as dendritic cells, macrophages, and B cells, as well as activated T cells. MHC-II molecules can present processed antigens to CD4+ T lymphocytes, which play a role in inducing immune response. Knocking out CIITA, RFX5, RFXAP, and/or RFXANK genes can significantly reduce MHC Class II molecule expression, thereby effectively evading T cell killing. The RFX complex can promote other transcription factors’ binding to the conserved sequences in the upstream regulatory regions of MHC-II genes, enhancing the binding specificity and affinity, and playing a role in the transcription of MHC-II genes.

HLA antigen genes are among the most polymorphic genes observed in the human genome. The generation of "universal donor" cells compatible with any HLA genotype offers a more affordable alternative strategy that could address immune rejection and immune evasion. To generate such allogeneic universal donor cell lines, functionally disrupting the expression of the MHC-I can be achieved, for example, by disrupting the expression of two genetic alleles encoding the MHC-I light chain β2M. However, it is envisioned that the resulting β2M-KO cell line and its derivatives will exhibit greatly reduced surface MHC class I molecules, and thus exhibit reduced immunogenicity to allogeneic CD8+ T cells, thereby effectively evading T cell killing.

However, to reduce the killing by NK cells as result of knocking out of the β2M gene, additional gene manipulation (e.g., making cells to overexpress a gene that antagonizes the killing activity of NK cells) are needed and may bring off-target risks. The present disclosure addresses this problem.

Reduced or eliminated RFX5 protein, RFXAP protein, and/or RFXANK protein

The present disclosure provides an approach to mitigating the HvGR in adoptive cell therapy via reducing or eliminating the expression or function of RFX5, RFXAP, RFXANK proteins or any combination thereof. Provided herein are cells with one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof. Cells with one or more such genomic alterations include progeny of cells that carry the same genomic alterations. In some cases, the cells are immune cells. In some cases, the cells are T cells, such as CD4+ and CD8+ T cells. In some cases, the cells are CAR-T cells (e.g., T cells expressing a CAR, not limited to CAR19 as described herein) . In some cases, the cells are allogeneic CAR-T cells. In some cases, the cells are isolated from cord blood. In some cases, the genomic alterations are achieved via CRISPR/Cas9 system described herein.

In some cases, the reduction or elimination of the expression or function of RFX5, RFXAP, RFXANK proteins or any combination thereof is achieved using CRISPR-Cas9 gene editing tools. The RFX5 gene can provide instructions for making a protein that primarily helps control the transcription of MHC class II genes.

Without wishing to be bound by a certain theory, RFX5 protein is part of a group of proteins named the regulatory factor X (RFX) complex. This complex can attach to a specific region of DNA involved in the regulation of MHC class II gene activity. The RFX5 protein can help the complex attach to the correct region of DNA. The RFX complex can attract other necessary proteins to this region and can help turn on MHC class II gene transcription, allowing production of MHC class II proteins.

Unexpectedly, data generated according to some embodiments of the present disclosure suggest that knockout of the RFX5 gene not only ablates MHC class II molecule expression but also ablates MHC class I molecules. In some embodiments, allogeneic immune cells with reduced or eliminated expression or function of RFX5 protein, RFXAP protein, RFXANK proteins or any combination thereof have reduced or eliminated expression of MHC class I molecules as compared to similar allogeneic immune cells with β2M gene knockout. In some cases, missing MHC class I molecules on the surface of allogeneic immune cells leads to killing of the allogeneic immune cells by NK cells of a subject receiving the allogeneic immune cells. In some embodiments, a number of allogeneic immune cells with reduced or eliminated expression or function of RFX5 protein, RFXAP protein, RFXANK proteins or any combination thereof that are killed by NK cells of a subject receiving the allogeneic immune cells is lower as compared to similar allogeneic immune cells with β2M gene knockout. In some cases, such genomic alteration (s) provides protection and improved viability of the allogeneic immune cells after administering to a subject.

In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFX5 protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFXAP protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFXANK protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFX5 protein and RFXAP protein, RFX5 protein and RFXANK protein, or RFXAP protein and RFXANK protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFX5 protein, RFXAP protein, and RFXANK protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that eliminate expression or function of RFX5 protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that eliminate expression or function of RFXAP protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that eliminate expression or function of RFXANK protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that eliminate expression or function of RFX5 protein and RFXAP protein, RFX5 protein and RFXANK protein, or RFXAP protein and RFXANK protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that eliminate expression or function of RFX5 protein, RFXAP protein and RFXANK protein.

In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce or eliminate expression of RFX5 gene, RFXAP gene, RFXANK gene, or any combination thereof. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFX5 gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFXAP gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFXANK gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFX5 and RFXAP gene, RFX5 and RFXANK gene, or RFXAP and RFXANK gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFX5, RFXAP and RFXANK protein. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that eliminate expression of RFX5 gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFXAP gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFXANK gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression of RFX5 and RFXAP gene, RFX5 gene and RFXANK gene, or RFXAP gene and RFXANK gene. In some cases, in a cell population provided herein, at least some of the cells comprise one or more genomic alterations that reduce expression or function of RFX5 protein, RFXAP protein and RFXANK protein.

In some cases, the one or more genomic alterations that reduce or eliminate expression of RFX5 gene, RFXAP gene, RFXANK gene, or any combination thereof comprises a knockout of RFX5 gene, RFXAP gene, RFXANK gene, or any combination thereof. In some cases, the one or more genomic alterations comprises a knockout of RFX5 gene. In some cases, the one or more genomic alterations comprises a knockout of RFXAP gene. In some cases, the one or more genomic alterations comprises a knockout of RFXANK gene. In some cases, the one or more genomic alterations comprises a knockout of RFX5 and RFXAP gene. In some cases, the one or more genomic alterations comprises a knockout of RFX5 and RFXANK gene. In some cases, the one or more genomic alterations comprises a knockout of RFXAP and RFXANK gene. In some cases, the one or more genomic alterations comprises a knockout of RFX5, RFXAP and RFXANK gene. In some cases, the one or more genomic alterations comprise a nonsense mutation. In some cases, the nonsense mutation is achieved by any genetic engineering or gene editing technology described in the present disclosure, for example, CRISPR/Cas9 system. In some cases, the one or more genomic alterations that reduce or eliminate expression of RFX5 gene, RFXAP gene, RFXANK gene, or any combination thereof comprises a nonsense mutation in RFX5 gene.

In various embodiments, at least some of the cells do not have knockout of β2M gene. In some cases, all of the cells do not have knockout of β2M gene. In various embodiments, at least some of the cells do not have genomic alterations that reduce or eliminate expression of β2M gene. In some embodiments, at least some of the cells do not have knockout of CIITA gene. In some cases, all of the cells do not have knockout of CIITA gene. In some embodiments, at least some of the cells do not have genomic alterations that reduce or eliminate expression of CIITA gene. In various embodiments, at least some of the cells do not have an exogenous nucleic acid sequence that encodes a NK inhibitory molecule. Exemplary NK inhibitory molecules include antibody or an antigen binding fragment thereof targeting an NK-inhibiting receptor. Exemplary NK-inhibiting receptor comprises NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM 1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4, and KLRG1. In some embodiments, the NK inhibitory molecule comprises a transmembrane domain, and a costimulatory domain.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16) , at least some of the cells in a cell population with one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof have reduced or eliminated expression and/or function of HLA-A, HLA-B, HLA-C, or HLA-DR protein, or combinations thereof. In some cases, at least some of the cells comprise a nonsense mutation in RFX5 gene. In some cases, the nonsense mutation is introduced to at least some of the cells CRISPR/Cas9 system (e.g., through a gRNA described herein) . In some cases, at least some of the cells express CAR (e.g., CAR19) .

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16) , at least some cells have reduced or eliminated expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof. In some cases, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%of the cells have reduced or eliminated expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16) , at least some cells have reduced or eliminated expression of RFX5 gene, RFXAP gene, RFXANK gene, or any combination thereof. In some cases, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%of the cells have reduced or eliminated expression of RFX5 gene, RFXAP gene, RFXANK gene, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-8) , at least some cells have reduced or eliminated expression or function of RFX5 protein. In some cases, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, at least about 50%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, at least about 60%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, at least about 70%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, at least about 80%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, at least about 90%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, at least about 95%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, about 100%of the cells have reduced or eliminated expression and/or function of RFX5 protein. In some cases, the one or more genomic alterations comprises knock out of RFX5 gene. In some cases, the one or more genomic alterations comprises a nonsense mutation in RFX5 gene.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFXANK protein (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 9-12) , at least some cells have reduced or eliminated expression or function of RFXANK protein. In some cases, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, at least about 50%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, at least about 60%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, at least about 70%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, at least about 80%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, at least about 90%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, at least about 95%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, about 100%of the cells have reduced or eliminated expression and/or function of RFXANK protein. In some cases, the one or more genomic alterations comprises knock out of RFXANK gene. In some cases, the one or more genomic alterations comprises a nonsense mutation in RFXANK gene.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFXAP protein (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 13-16) , at least some cells have reduced or eliminated expression or function of RFXAP protein. In some cases, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, at least about 50%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, at least about 60%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, at least about 70%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, at least about 80%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, at least about 90%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, at least about 95%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, about 100%of the cells have reduced or eliminated expression and/or function of RFXAP protein. In some cases, the one or more genomic alterations comprises knock out of RFXAP gene. In some cases, the one or more genomic alterations comprises a nonsense mutation in RFXAP gene.

In some cases, after contacting with reagents that induce the reduced or eliminated expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16) , at least some cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 40%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 50%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 55%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 60%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 65%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 70%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 75%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 80%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 85%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 90%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 95%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein. In some cases, at least about 99%of the cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, or HLA-C protein.

In some cases, after contacting with reagents that induce the reduced or eliminated expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16) , at least some cells have reduced or eliminated expression and/or function of HLA-DR protein. In some cases, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 50%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 55%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 60%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 65%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 70%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 75%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 80%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 85%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 90%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 95%of cells in the population have reduced or eliminated expression or function of HLA-DR protein. In some cases, at least about 99%of cells in the population have reduced or eliminated expression or function of HLA-DR protein.

In some cases, after contacting with reagents that induce the reduced or eliminated expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16) , at least some cells have reduced or eliminated expression and/or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 50%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 55%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 60%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 65%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 70%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 75%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 80%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 85%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 90%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 95%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins. In some cases, at least about 99%of cells in the population have reduced or eliminated expression or function of HLA-A, HLA-B, HLA-C, and HLA-DR proteins.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16, such as gRNA having a sequence of SEQ ID NO: 7) and co-culturing with allogeneic NK cells (e.g., after 1, 2, 3, 4, 5 or 6 days at an effector-target ratio of about 1: 1, 2: 1, or 4: 1) , the cells proliferate at a rate that is at least about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1.0-fold, 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, or about 2.0-fold relative to that of a reference population of cells, wherein the reference population of cells is substantially the same as the population of cells except the reference population of cells has knockout of β2M gene and does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16, such as gRNA having a sequence of SEQ ID NO: 7) and co-culturing with autologous NK cells (e.g., after 1, 2, 3, 4, 5 or 6 days at an effector-target ratio of about 1: 1, 2: 1, or 4: 1) , the cells proliferate at a rate that is at least about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1.0-fold, 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, or about 2.0-fold relative to that of a reference population of cells, wherein the reference population of cells is substantially the same as the population of cells except the reference population of cells has knockout of β2M gene and does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16, such as gRNA having a sequence of SEQ ID NO: 7) and co-culturing with autologous NK cells (e.g., after 1, 2, 3, 4, 5 or 6 days at an effector-target ratio of about 1: 1, 2: 1, or 4: 1) , a percentage of the cells that are CD4+ and ABC-is at least about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1.0-fold, 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, or about 2.0-fold relative to that of a reference population of cells, wherein the reference population of cells is substantially the same as the population of cells except the reference population of cells has knockout of β2M gene and does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16, such as gRNA having a sequence of SEQ ID NO: 7) and co-culturing with autologous NK cells (e.g., after 1, 2, 3, 4, 5 or 6 days at an effector-target ratio of about 1: 1, 2: 1, or 4: 1) , a percentage of the cells that are CD8+ and ABC-is at least about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1.0-fold, 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, or about 2.0-fold relative to that of a reference population of cells, wherein the reference population of cells is substantially the same as the population of cells except the reference population of cells has knockout of β2M gene and does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16, such as gRNA having a sequence of SEQ ID NO: 7) and co-culturing with allogeneic NK cells (e.g., after 1, 2, 3, 4, 5 or 6 days at an effector-target ratio of about 1: 1, 2: 1, or 4: 1) , a percentage of the cells that are CD4+ and ABC-is at least about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1.0-fold, 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, or about 2.0-fold relative to that of a reference population of cells, wherein the reference population of cells is substantially the same as the population of cells except the reference population of cells has knockout of β2M gene and does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof (e.g., comprising gRNA having a sequence of any one of SEQ ID NOs: 4-16, such as gRNA having a sequence of SEQ ID NO: 7) and co-culturing with allogeneic NK cells (e.g., after 1, 2, 3, 4, 5 or 6 days at an effector-target ratio of about 1: 1, 2: 1, or 4: 1) , a percentage of the cells that are CD8+ and ABC-is at least about 0.7-fold, about 0.8-fold, about 0.9-fold, about 1.0-fold, 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, or about 2.0-fold relative to that of a reference population of cells, wherein the reference population of cells is substantially the same as the population of cells except the reference population of cells has knockout of β2M gene and does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after co-culturing with target cells that comprise a surface antigen that is recognized by the CAR (e.g., for 72 hours at an effector-target ratio of 1: 1, 1: 3, or 1: 8) , a percentage of cells expressing CD107a is from about 70%to about 110%, from about 70%to about 80%, from about 80%to about 90%from about 90%to about 100%, from about 100%to about 110%, from about 70%to about 100%, from about 70%to about 90%, from about 80%to about 110%, from about 80%to about 100%, or from about 90%to about 110%relative to that of a reference population of cells, wherein the reference population of cells is same as the population of cells except the reference population of cells does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof.

In some cases, after co-culturing with target cells (e.g., Raji cells) that comprise a surface antigen that is recognized by the CAR (e.g., for 72 hours at an effector-target ratio of 1: 1, 1: 3, or 1: 9) , the cells cause lysis of at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%of target cells relative to that of a reference population of cells, wherein the reference population of cells is same as the population of cells except the reference population of cells does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof. In some cases, the cells and the reference population of cells are CD8+. In some cases, the cells and the reference population of cells are CD4+.

In some cases, after co-culturing with target cells (e.g., Raji cells) that comprise a surface antigen that is recognized by the CAR (e.g., for 72 hours at an effector-target ratio of 1: 1, 1: 3, or 1: 9) , a percentage of cells expressing CD107a is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%relative to that of a reference population of cells, wherein the reference population of cells is same as the population of cells except the reference population of cells does not have the one or more genomic alterations that reduce or eliminate expression and/or function of RFX5 protein, RFXAP protein, RFXANK protein, or any combination thereof. In some cases, the percentage of cells expressing CD107a is at least about 70%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at least about 70%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at least about 75%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at least about 80%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at least about 85%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at least about 90%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at least about 95%relative to that of a reference population of cells. In some cases, the percentage of cells expressing CD107a is at about 100%relative to that of a reference population of cells. In some cases, the cells and the reference population of cells are CD8+. In some cases, the cells and the reference population of cells are CD4+.

Reducing Graft-Versus-Host Disease (GvHD)

The present disclosure provides, in some aspects, compositions, methods, and kits related to allogeneic adoptive cell therapy with reduced or eliminated GvHD. GvHD can occur after allogeneic immune cells are administered to a recipient (e.g., a subject in need thereof) , eliciting an immune response that can result in host tissue damage. Mediators of GvHD can include αβ T cells. The major histocompatibility complex (MHC) on the membrane of the cell of the subject receiving the immune cell therapy can present antigen peptide to the αβ T cell receptors (TCRαβ) on the allogeneic immune cell membrane, which can induce the allogeneic T cells to attack the subject’s normal cells or tissues. In some cases, T cell receptors (TCR) on allogeneic T cells can specifically recognize the antigen peptide-MHC molecular complex presented by antigen presenting cells (APC) or target cells, thereby identifying and killing normal cells in patients to result in toxic effects. TCR is a dimer molecule comprising α and βchains. TCRαβ can comprises a TCRα chain, encoded by a single TRAC gene, complexed with a TCRβchain, encoded by two TRBC genes. A complete disruption can be achieved by knockout of the TRBC gene, since the TCRαβ dimer is necessary for full function of TCR. TCR can bind to CD3γ, ε, δ and ζchains involved in signal transduction to form TCR-CD3 complexes, which are jointly involved in the recognition of antigens by T cells. CD3 expression cannot be detected on the cell membrane after TCR knockout. The expression of surface CD3 molecules (CD3 molecules expressed on cell membrane) can be detected by flow cytometry to reflect the expression of TCR molecules. In some case, the CRISPR/Cas9 system with electroporation is used for delivery of a RNP described herein (e.g., comprising gRNA with a sequence of SEQ ID NO: 1) to achieve TRBC gene knockout. In some cases, cells are sorted using CD3 MicroBeads to obtain cells with a TCR-ratio of about 99.9%.

The present disclosure provides, in some aspects, an approach to reducing or eliminating GvHD via reducing or eliminating expression and/or function of a component of a T cell receptor (TCR) /CD3 complex of the allogeneic immune cells. In some cases, the reducing or eliminating expression and/or function of a component of a T cell receptor (TCR) /CD3 complex is achieved using CRISPR-Cas9 gene editing tools. Without wishing to be bound by a certain theory, immune cells with reduced or eliminated TCRαβ expression on their surface can cause very low or no alloreactive immune response against normal tissues of the recipient. TCRαβ complexes with CD3 molecules on the T cell surface to elicit immune responses. Without wishing to be bound by a certain theory, T cells with reduced or eliminated expression of a TCR component also have reduced or loss of surface CD3 expression and capabilities for activation via either the CD3 complex or through the TCR. In some cases, allogeneic immune cells provided herein do not comprise a genomic alteration that affects the TRAC gene.

Reduced or eliminated component of a T cell receptor (TCR) /CD3 complex

The present disclosure provides an approach to mitigating the GvHD in adoptive cell therapy via reducing or eliminating the expression or function of a component of a T cell receptor (TCR) /CD3 complex. Provided herein are cells with one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex. Cells with one or more such genomic alterations include progeny of cells that carry the same genomic alterations. Cells with one or more such genomic alterations include progeny of cells that carry the same genomic alterations. In some cases, the cells are immune cells. In some cases, the cells are T cells, such as CD4+and CD8+ T cells. In some cases, the cells are CAR-T cells. In some cases, the cells are allogeneic CAR-T cells. In some cases, the cells are isolated from cord blood. In some cases, the genomic alterations are achieved via CRISPR/Cas9 system described herein.

In some cases, the component of a TCR/CD3 complex is selected from the group consisting of: TCR α subunit constant region, TCR β subunit constant region, CD3ζ, CD3γ, CD3δ, CD3ε, and combinations thereof. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of TCR α subunit constant region. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of TCR β subunit constant region. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3ζ. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3γ. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3δ. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3ε. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of TCR α subunit constant region and TCR β subunit constant region. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3ζ and TCR β subunit constant region. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3γ and TCR βsubunit constant region. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3δ and TCR β subunit constant region. In some cases, at least some of the cells have one or more genomic alterations that reduce or eliminate expression and/or function of CD3ε and TCR β subunit constant region.

In some cases, the one or more genomic alterations that reduces or eliminates expression and/or function of a component of a T cell receptor (TCR) /CD3 complex comprises one or more genomic alterations that reduce or eliminate expression of TRAC gene, TRBC gene, CD247 gene, CD3G gene, CD3D gene, CD3E gene, and combinations thereof. In some cases, the one or more genomic alterations comprise knockout of TRAC gene. In some cases, the one or more genomic alterations comprise knockout of TRBC gene. In some cases, the one or more genomic alterations comprise knockout of CD247 gene. In some cases, the one or more genomic alterations comprise knockout of CD3G gene. In some cases, the one or more genomic alterations comprise knockout of CD3D gene. In some cases, the one or more genomic alterations comprise knockout of CD3E gene. In some cases, the one or more genomic alterations comprise knockout of TRAC gene and TRBC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in TRAC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in TRBC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD247 gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD3G gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD3D gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD3E gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in TRAC gene and TRBC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD247 gene and TRBC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD3G gene and TRBC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD3D gene and TRBC gene. In some cases, the one or more genomic alterations comprise a nonsense mutation in CD3E gene and TRBC gene. In some cases, the one or more genomic alterations are achieved via CRISPR/Cas9 system.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex (e.g., comprising gRNA having a sequence of SEQ ID NO: 1) , at least some of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9%of the cells have knockout of TRBC gene. In some cases, at least about 80%of the cells have knockout of TRBC gene. In some cases, at least about 85%of the cells have knockout of TRBC gene. In some cases, at least about 90%of the cells have knockout of TRBC gene. In some cases, at least about 95%of the cells have knockout of TRBC gene. In some cases, at least about 96%of the cells have knockout of TRBC gene. In some cases, at least about 97%of the cells have knockout of TRBC gene. In some cases, at least about 98%of the cells have knockout of TRBC gene. In some cases, at least about 99%of the cells have knockout of TRBC gene. In some cases, at least about 99.5%of the cells have knockout of TRBC gene. In some cases, at least about 99.9%of the cells have knockout of TRBC gene. In some cases, at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 80%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 85%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 90%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 95%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 96%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 97%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 98%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 99%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 99.5%of the cells have a nonsense mutation in TRBC gene. In some cases, at least about 99.9%of the cells have a nonsense mutation in TRBC gene.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex (e.g., comprising gRNA having a sequence of SEQ ID NO: 1) , at least some of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 80%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 85%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 90%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 95%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 96%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 97%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 98%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least about 99%of the cells have reduced expression of TCRβsubunit on the cell membrane. In some cases, at least about 99.5%of the cells have reduced expression of TCRβ subunit on the cell membrane. In some cases, at least some of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 80%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 85%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 90%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 95%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 96%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 97%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 98%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 99%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 99.5%of the cells have reduced expression of TCRαβ dimer on the cell membrane.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex (e.g., comprising gRNA having a sequence of SEQ ID NO: 1) , at least some of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 80%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 85%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 90%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 95%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 96%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 97%of the cells have eliminated expression of TCRαβdimer on the cell membrane. In some cases, at least about 98%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 99%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least about 99.5%of the cells have eliminated expression of TCRαβ dimer on the cell membrane. In some cases, at least some of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 80%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 85%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 90%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 95%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 96%of the cells have reduced expression of TCRαβdimer on the cell membrane. In some cases, at least about 97%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 98%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 99%of the cells have reduced expression of TCRαβ dimer on the cell membrane. In some cases, at least about 99.5%of the cells have reduced expression of TCRαβ dimer on the cell membrane.

In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex (e.g., comprising gRNA having a sequence of SEQ ID NO: 1) , at least some of the cells have reduced or eliminated expression of CD3 on the cell membrane. In some cases, at least some of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least about 70%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least about 75%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least about 80%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least about 85%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least or about 90%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least about 95%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least about 99%of the cells have reduced expression of CD3 on the cell membrane. In some cases, at least some of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least about 70%of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least about 75%of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least about 80%of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least about 85%of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least or about 90%of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least about 95%of the cells have eliminated expression of CD3 on the cell membrane. In some cases, at least about 99%of the cells have eliminated expression of CD3 on the cell membrane.

In some cases, anti-CD3/anti-CD28 beads can be employed to mimic the stimulation of MHC antigen peptides to measure the ability of the cells to recognize MHC antigen peptides or mediate GvHD. In some cases, after contacting with reagents that induce the one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex (e.g., comprising gRNA having a sequence of SEQ ID NO: 1) , less than about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%of the cells are proliferating after contacting with anti-CD3/anti-CD28 beads (e.g., for 3 days at a bead : cell ratio of about 0: 1, 0.5: 1, 1: 1, 2: 1) . In some cases, the cell proliferation can be measure by Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling.

Also provided herein is a method of reducing or eliminating the expression or function of a component of a T cell receptor (TCR) /CD3 complex in a cell (e.g., to form the immune cell or CAR-T described herein) or a group of cells (e.g., to form a population of immune cells or CAR-T cells with genomic alteration (s) ) . Also provided herein is a method of reducing or eliminating the expression or function of TCRβ subunit, TCRαβ dimer, and/or CD3 on the cell membrane protein in a cell (e.g., to form the immune cell or CAR-T described herein) or a group of cells (e.g., to form a population of immune cells or CAR-T cells with genomic alteration (s) ) . The methods comprise contacting the cell or group of cells with reagents that induce one or more genomic alterations that reduce or eliminate expression and/or function of a component of a T cell receptor (TCR) /CD3 complex (e.g., comprising gRNA having a sequence of SEQ ID NO: 1) .

Immune Cells of the Present Disclosure

In one aspect, provided herein is an immune cell that (i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein, and (ii) does not have knockout of β2M gene, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and/or HLA-C gene. In some cases, the one or more genomic alterations comprise one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene. In some cases, the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene comprise deletion, insertion, substitution, or any combination thereof. Also provided herein is an immune cell that (i) has a genomic alteration that reduces or eliminates expression or function of one or more of RFX5, RFXAP, or RFXANK gene, and (ii) does not have knockout of β2M gene, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and/or HLA-C gene. In some cases, the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene. In some embodiments, the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene. In some embodiments, the immune cell does not have knockout of β2M gene. In some embodiments, immune cell does not have knockdown of β2M gene. In some embodiments, the immune cell does not have both knockout and knockdown of β2M gene.

In some embodiments, at least 70%of the immune cells in the population have a genomic

In some embodiments, the immune cell is a lymphocyte or a myeloid cell.

In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell. In some embodiments, the lymphocyte is a CD8+ T cell or a CD4+ T cell. In some embodiments, the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .

In some embodiments, the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some embodiments, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε. In some cases, the T cell has a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof. The TCR/CD3 complex component can be encoded by TRAC, TRBC, CD247, CD3G, CD3D, CD3E gene, or any combination thereof.

In some embodiments, the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a TCR/CD3 gene. The TCR/CD3 gene can be TRAC, TRBC, CD247, CD3G, CD3D, CD3E genes, or any combination thereof.

In some embodiments, the immune cell is a myeloid cell. The term “myeloid cell” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from myeloid progenitors including tissue specific and specialized varieties, and encompasses, by way of non- limiting example, granulocytes (i.e., mast cells, neutrophils, eosinophils and basophils) , monocytes, macrophages, and dendritic cells.

In some embodiments, the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord.

In some embodiments, the immune cell is derived from an autologous donor or an allogenic donor. In some embodiments, the immune cell is derived from an allogenic donor.

In some embodiments, the immune cell does not have knockout or does not have knockdown of a NK activating receptor ligand gene. In some embodiments, the immune cell does not have knockout of a NK activating receptor ligand gene. In some embodiments, the immune cell does not have knockdown of a NK activating receptor ligand gene. In some embodiments, the immune cell does not have knockout or knockdown of a NK activating receptor ligand gene. Exemplary NK activating receptor ligand genes include MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60, MULT1, B7-H6, BAG6, PfEMP1, HSPGS, AICL, CD112, CD155, CD48, CD58, CD59, ICAM1, ICAM2, ICAM3, STAT1, JAK1, IFNGR2, JAK2, or IFNGR 1.

In some embodiments, the immune cell does not have a heterologous nucleic acid encoding a NK inhibitory molecule. Exemplary NK inhibitory molecules include antibody or an antigen binding fragment thereof targeting an NK-inhibiting receptor, wherein the NK-inhibiting receptor is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM 1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4, and KLRG1. In some embodiments, the NK inhibitory molecule comprises a transmembrane domain, and a costimulatory domain.

In some embodiments, the immune cell has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to a corresponding immune cell that has knockout of β2M gene, e.g., an increase by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more. In some embodiments, the corresponding immune cell does not have one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein. In some embodiments, the corresponding immune cell does not have a genomic alteration that reduces or eliminates expression or function of one or more of RFX5, RFXAP, or RFXANK protein. In some embodiments, the NK cell-mediated cellular cytotoxicity is measured by an assay in which the immune cell is contacted with NK cells. In some embodiments, the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord.

In some embodiments, the immune cell has comparable (e.g., about 100%, about 100%±5%, about 100%±10%, about 100%±15%, or about 100%±20%) tolerance to T cell-mediated cellular cytotoxicity, as compared to the corresponding immune cell.

In another aspect, provided herein is a T cell comprising a chimeric antigen receptor (CAR) (CAR-T cell) , wherein the CAR-T cell (i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein, (ii) does not have knockout of β2M gene, and (iii) has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some embodiments, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.

In another aspect, provided herein is a T cell comprising a chimeric antigen receptor (CAR) (CAR-T cell) , wherein the CAR-T cell (i) has a genomic alteration that reduces or eliminates expression or function of one or more of RFX5, RFXAP, or RFXANK protein, (ii) does not have knockout of β2M gene, and (iii) has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some embodiments, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε. In some embodiments, the TCR/CD3 complex component is encoded by one or more genes selected from TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.

In another aspect, provided herein is a T cell comprising a chimeric antigen receptor (CAR) (CAR-T cell) , wherein the CAR-T cell (i) has a genomic alteration that reduces or eliminates expression or function of one or more of RFX5, RFXAP, or RFXANK gene, (ii) does not have knockout of β2M gene, and (iii) has a genomic alteration that reduces or eliminates expression or function of a TCR/CD3 gene. In some embodiments, the TCR/CD3 gene is one or more selected from TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.

In some embodiments, the CAR-T cell is derived from a helper T cell, a cytotoxic T cell, a memory T ceil, regulatory T cell, gamma delta T cell, a natural killer T cell, cytokine induced killer cell, a T memory stem cell, or other T effector cell. It can be also useful for the T cell to have limited toxicity toward healthy cells.

In some embodiments, the T cell is an autologous cell. In another embodiment, the T cell is an allogeneic cell. In some embodiments, the T cells are primary cells, expanded T cells derived from primary T cells, T cells derived from stem cells differentiated in vitro, T cell lines such as Jurkat cells, other sources of T cells, or any combinations thereof.

As used here in, a “chimeric antigen receptor” ( “CAR” ) refer to a set of polypeptides that may comprise an extracellular domain (e.g., an antigen binding domain) that binds specifically to a target, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain may comprise a costimulatory signaling region. The CAR can also comprise a signal peptide or a leader sequence covalently joined to the N-terminus of the extracellular domain.

The extracellular domain. The extracellular domain that binds to a specific target (e.g., a cancer cell, a tumor-specific antigen, or a tumor-associated antigen) can be an antibody or any fragments thereof. In certain embodiments, the extracellular domain/antigen binding domain may comprise a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) , and a light chain variable region comprising three light chain complementarity determining regions (LCDRs) . In some embodiment, the extracellular domain is a Fab or a scFv. In some instances, it is beneficial that the antigen binding domain is derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it can be beneficial that the antigen binding domain of the CAR may comprise a human or humanized antibody or a fragment thereof that binds a human antigen. In one exemplary embodiment, the CAR may bind a target in a mammal (e.g., a human) .

Transmembrane domain and/or Hinge Domain. With respect to the transmembrane domain, in various embodiments, the CAR comprises a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the CAR may comprise a transmembrane domain that naturally is associated with one of the domains in the CAR. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize interactions with other members of the receptor complex.

The transmembrane domain can be derived either from a natural or from a synthetic source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one embodiment, the transmembrane domain is synthetic, in which case it may comprise predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain. Optionally, a short oligo-or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine (GS) doublet provides a particularly suitable linker.

In some embodiments, a variety of human hinges can be employed as well, including, but not limited to, the human Ig (immunoglobulin) hinge domain and the CD8 alpha hinge domain. Examples of the hinge and/or transmembrane domain include, but are not limited to, a hinge and/or transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIR, 0X40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRFl) , CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, IT GAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D) , SLAMF6 (NTB-A, Lyl08) , SLAM (SLAMFl, CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C.

Intracellular Signaling Domain. The CAR of the present disclosure may comprise an intracellular signaling domain, wherein the intracellular signaling domain may comprise a costimulatory signaling region. The intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed. The intracellular domain transduces the effector function signal and directs the cell to perform its specialized function. Examples of an intracellular signaling domain include, but are not limited to, the cytoplasmic portion of a surface receptor, a co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

As used herein, a “costimulatory molecule, ” refers to a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Exemplary costimulatory molecules including but are not limited to CD27, CD28, 4-1BB (CD137) , OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand that specifically binds with CD83.

Examples of the intracellular signaling domain include, without limitation, the ζ chain of the T cell receptor complex or any of its homologs, e.g., η chain, FcsRFγ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε) , syk family tyrosine kinases (Syk, ZAP70, etc. ) , src family tyrosine kinases (Lck, Fyn, Lyn, etc. ) , and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain is human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof. In certain embodiments, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS (CD278) , 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof. In certain embodiments, the intracellular domain may comprise a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-IBB (CD 137) , OX40 (CD134) , PD-1, CD7, LIGHT, CD83L, DAPIO, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276) , or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR) .

The disclosure is not limited to a specific CAR. Rather, any CAR that targets any antigen of interest, can be used in the present disclosure. For example, the CAR of the present disclosure may specifically bind to a tumor-specific antigen. The tumor-specific antigen can be a molecule, including but not limited to a protein, polypeptide, peptide, lipid, carbohydrate, etc., predominantly expressed or over-expressed by a tumor cell, such that the antigen can be regarded as specifically associated with the tumor or cancer. The tumor-specific antigen can be expressed by normal, non-tumor, or non-cancerous cells but a level that is lower or not as robust as the expression by tumor cells. The tumor cells can over-express the tumor-specific antigen or express the tumor-specific antigen at a significantly higher level than that by normal, non-cancerous cells. The tumor-specific antigen can be expressed by cells of a different state of development or maturation. For example, the tumor-specific antigen can be expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult subject. The tumor-specific antigen can be expressed by stem cells or precursor cells, which are not normally found in an adult subject. In some cases, tumor-specific antigen can be a mutated antigen that is predominantly expressed or overexpressed by tumor or cancer cells and not expressed or expressed at a significantly lower level by normal, non-cancerous cells. In some embodiments, the tumor-specific antigen is CD19. CD19-specific CAR molecule (or “CAR19” as used herein interchangeably) can be expressed in CAR-T cells by transducing the T cells (e.g., cord blood-derived T cells) with the 3rd generation replication-deficient lentivirus (LV) carrying the gene of interest (e.g., CAR19) .

ACAR molecule can comprise an extracellular CD19 antigen binding domain, a CD8 spacer or hinge region, a CD8 transmembrane domain and a CD3 ζ intracellular signaling domain. A CAR can comprise a ligand binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. The antigen-binding domain can comprise a variable regions of antibody heavy (VH) and light (VL) chains connected via a flexible linker to form a single chain fragment variable (scFv) and to determine the binding specificity. In some cases, the scFv used in the present disclosure is from FMC63 mouse hybridoma. Exemplary scFv is described in U.S. Pat. Nos. 9,540,445, 10,357,514, and 11,384,155; U.S. Pat. Publication No. US20210061910; each of which is hereby incorporated by reference. A hinge region can be the spacer region that exposes the antigen-binding domain on CAR-T cell surface for antigens binding. In some cases, a hinge region is from the CD8 transmembrane domain. The signaling of CARs can be transduced by the intracellular signaling domains, CD3ζ chain (activation) and 4-1BB (co-stimulatory) , at the C-terminal end.

In some embodiments, the ligand binding domain is a scFv fragment. In some embodiments, the ligand binding domain targets CD19. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 75%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 85%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 90%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 95%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 98%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is at least 99%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain comprises an amino acid sequence that is 100%identical to the sequence set forth in SEQ ID NO: 20. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 75%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 85%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 90%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 95%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 98%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is at least 99%identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the ligand binding domain is encoded by a nucleic acid sequence that is 100%identical to the sequence set forth in SEQ ID NO: 19.

In some cases, CAR comprises a leader. The leader can locate at the N-terminal of the CAR. The leader can be connected to the ligand binding domain. In some embodiments, the leader comprises the leader of CD8α. In some embodiments, the leader comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence of SEQ ID NO: 18. In some embodiments, the leader is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence of SEQ ID NO: 17.

In some cases, CAR comprises a hinge region. The hinge region can connect the ligand binding domain and the transmembrane domain. In some cases, the hinge region is from a human protein. In some embodiments, the hinge region comprises hinge region of human Ig hinge, such as IgG1 IgG4, IgD, FcγRIIIα, a KIR2DS2 hinge, or CD8α hinge. In some embodiments, the hinge region comprises a flexible linker described herein, for example, a GS linker. In some embodiments, the hinge region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 22. In some embodiments, the hinge region is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 21.

The transmembrane domain can be derived from the transmembrane domain of TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, or any combination thereof. In some embodiments, the transmembrane domain comprises a transmembrane domain of TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, or any fragments thereof.

In some embodiments, the co-stimulatory domain is a co-stimulatory domain of CD28. In some embodiments, the co-stimulatory domain is a co-stimulatory domain of CD137 (4-1BB) . In some embodiments, the co-stimulatory domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 26. In some embodiments, the co-stimulatory domain is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 25.

In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8α or fragments thereof. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 24. In some embodiments, the transmembrane domain is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 23.

The intracellular signaling domain can comprise at least a portion of an intracellular signaling domain from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 28. In some embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 27.

In some embodiments, the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 75%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 85%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 90%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 95%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 98%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is at least 99%identical to the sequence set forth in SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence that is 100%identical to the sequence set forth in SEQ ID NO: 30.

In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 75%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 85%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 90%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 95%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 98%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 99%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the CAR is encoded by a nucleic acid sequence that is 100%identical to the sequence set forth in SEQ ID NO: 29.

Also provided herein are cells comprising an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) . In some cases, the cells are immune cells. In some cases, the cells are T cells, such as CD4+ and CD8+ T cells. In some cases, the cells are CAR-T cells. In some cases, the cells are allogeneic CAR-T cells. In some cases, the cells are isolated from cord blood. In some cases, at least some of the cells comprise the exogenous nucleic acid sequence encoding the CAR (e.g., CAR19) . In some cases, at least about 80%, about 85%, about 90%, or about 95%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 80%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 85%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 90%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 95%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 96%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 96%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 97%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 98%of the cells express the CAR (e.g., CAR19) . In some cases, at least about 99%of the cells express the CAR (e.g., CAR19) . In some cases, about 100%of the cells express the CAR (e.g., CAR19) .

In some embodiments, the CAR-T cells binds to GRAMD1A, KCNK3, RAI2, NPL, STC1, TOM1, F3, SLC6A8, SLC22A4, SERINC3, DDIT4L, LY96, NFASC, IFNGR1, DNER, SLC22A1, ITGB3, LRP10, ICAM1, ULBP2, SLC22A15, APLPl, ABTB2, AFF1, AGPAT2, AGTRAP, AKAP6, BFSP1, BHLHE40, CARD6, CCDC69, CCDC71L, FAM219A, FAM219B, FAM43A, FAM8A1, FOLR3, GSAP, GYS1, HECW2, HIF1A, INHBA, MAP3K8, MT-ND5, MT-ND6, and PRICKLE2. Other examples of such additional targets include, but are not limited to LRP12, SLC6A8, ITGB3, LRP10, BTN2A2, ICAM1, ABCAl, SLC22A23, TMEM63B, SLC37A1, SLC22A4, ENPP4, VNN1, SERINC3, ITGA11, SERINC2, ULBP2, SLC22A15, APLPl, DPP4, ABC A3, TPCN1, ABTB2, AFF1, AGPAT2, AGTRAP, AHNAK2, AK4, AKAP6, ALS2CL, AMPD3, ANKRD1, ANKRD29, ANKRD42, AOX1, ARHGEF37, ARRDC4, ATP6V1H, BFSP1, BHLHE40, BHLHE41, BTG2, C3, CARD6, CASP4, CCDC69, CCDC71L, CDKN1A, CHST15, COQIOB, CPPED1, CTSB, CYB5R1, CYBA, CYFIP2, CYP26B1, DDIT4L, DIRC3, DNAJB9, DTX4, DYNLT3, ELL2, ELOVL7, EML1, FADS3, FAM210B, FAM219A, FAM219B, FAM43A, FAM8A1, FILIP 1L, FOLR3, FOXOl, GFPT2, GM2A, GPX3, GRAMD1A, GRBIO, GSAP, GYS1, HECW2, HIF1A, HIST2H2BE, IDS, IGFN1, INHBA, JUN, KCNJ15, KCNK3, KDM6B, KIAA1217, KLHL21, LCP1, LINC00862, LY96, LYPLALl, LZTS3, MAP1LC3B, MAP3K10, MAP3K8, MAP7, MAPRE3, MAST3, MOAPl, MSC, MT-ND3, MT-ND5, MT-ND6, MXD1, MYOID, NABPl, NOV, NPL, OGFRLl, P4HA2, PGM2L1, PHYH, PLA2G15, PLA2G4C, PLD1, PLEKHG5, PLOD2, PPARGCIA, PPP2R5B, PRICKLE2, PSAP, RAB29, RAB36, RAB6B, RAG1, RAI2, RETSAT, RIOK3, RNF11, RNF14, RSPH3, RUSC2, SAT1, SCG5, SEL1L3, SERPINI1, SESN2, SIAE, SOD2, SPATA18, SPTBN2, SRPX2, ST20-AS1, STC1, STK38L, STON2, SUSD6, TAF13, TAPI, TBC1D2, TFEC, TNFAIP3, TNFAIP8L3, TOM1, TPRG1L, TSKU, TTC9, TXNIP, UBA6-AS1, VPS 18, WDR78, ZFHX2, or ZNFX1.

Compositions and methods of making and using CARs are well known in the art. See, for example, PCT/US2011/064191, which is incorporated by reference in its entirety herein.

Immune Cell Populations of the Present Disclosure

In another aspect, the present disclosure provides a population of immune cells resulted from genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, wherein at least 70%of immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene. The present disclosure also provides a population of immune cells resulted from genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK gene, wherein at least 70%of immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene. In some embodiments, the at least 70%of the immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene.

In some embodiments, the genetic engineering does not knock out β2M gene. In some embodiments, the population of immune cells does not have knockout of β2M gene. In some embodiments, the population of immune cells does not have knockdown of β2M gene. In some embodiments, the population of immune cells does not have both knockout and knockdown of β2M gene.

In some embodiments, at least 70%of the immune cells in the population have a genomic alteration that reduces or eliminates the expression or function of the one or more of RFX5, RFXAP, or RFXANK gene. In some embodiments, at least 70%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of the one or more of RFX5, RFXAP, or RFXANK protein. In some embodiments, at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have a genomic alteration that reduces or eliminates the expression or function of one or more of RFX5, RFXAP, and RFXANK gene. In some embodiments, at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of one or more of RFX5, RFXAP, and RFXANK protein. In some embodiments, at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene. In some cases, the one or more genomic alterations comprise deletion, insertion, substitution, or any combination thereof. In some cases, the one or more genomic alterations result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene.

In some embodiments, the population of immune cells comprises lymphocytes or myeloid cells.

In some embodiments, the immune cell of the population of immune cells is a lymphocyte. In some embodiments, the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell. In some embodiments, the lymphocyte is a CD8+ T cell or a CD4+ T cell. In some embodiments, the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .

In some embodiments, the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a TCR/CD3 complex component. The TCR/CD3 complex component can be encoded by TRAC, TRBC, CD247, CD3G, CD3D, CD3E genes, or any combination thereof.

In some embodiments, the immune cell of the population of immune cells is a myeloid cell. The term “myeloid cell” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from myeloid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, granulocytes (i.e., mast cells, neutrophils, eosinophils and basophils) , monocytes, macrophages, and dendritic cells.

In some embodiments, the immune cell of the population of immune cells is derived from peripheral blood, bone marrow, placenta, or umbilical cord.

In some embodiments, the immune cell of the population of immune cells is derived from an autologous donor or an allogenic donor. In some embodiments, the immune cell of the population of immune cells is derived from an autologous donor. In some embodiments, the immune cell of the population of immune cells is derived from an allogenic donor.

In some embodiments, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not knock out a NK activating receptor ligand gene in the immune cell. In some embodiments, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not knock out a NK activating receptor ligand gene in the immune cell.

In some embodiments, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK gene does not knock out a NK activating receptor ligand gene in the immune cell. In some embodiments, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not knock out a NK activating receptor ligand gene in the immune cell.

In some embodiments, the population of immune cells does not have knockout or does not have knockdown of a NK activating receptor ligand gene. In some embodiments, the population of immune cells does not have knockout or knockdown of a NK activating receptor ligand gene. In some embodiments, the population of immune cells does not have knockout of a NK activating receptor ligand gene. In some embodiments, the population of immune cells does not have knockdown of a NK activating receptor ligand gene. Exemplary NK activating receptor ligand genes include MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60, MULT1, B7-H6, BAG6, PfEMP1, HSPGS, AICL, CD112, CD155, CD48, CD58, CD59, ICAM1, ICAM2, ICAM3, STAT1, JAK1, IFNGR2, JAK2, or IFNGR1.

In some embodiments, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cells. In some embodiments, the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK gene does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cells. Exemplary NK inhibitory molecules include antibody or an antigen binding fragment thereof targeting an NK-inhibiting receptor, wherein the NK-inhibiting receptor is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM 1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4, and KLRG1. In some embodiments, the NK inhibitory molecule comprises a transmembrane domain, and a costimulatory domain.

In some embodiments, the population of immune cells does not have a heterologous nucleic acid encoding a NK inhibitory molecule.

In some embodiments, the percentage of the immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in the population is greater than the percentage of immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in a population of corresponding immune cells resulted from genetic engineering to knock out β2M gene. In some embodiments, the genetic engineering to knock out β2M gene does not knock out one or more of RFX5, RFXAP, or RFXANK gene. In some embodiments, the population of corresponding immune cells does not have knockout of one or more of RFX5, RFXAP, or RFXANK gene.

In some embodiments, the population of immune cells has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to the population of corresponding immune cells that does not have knockout of one or more of RFX5, RFXAP, or RFXANK gene, e.g., an increase by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more. In some embodiments, the NK cell-mediated cellular cytotoxicity is measured by an assay in which the population of immune cells is contacted with NK cells. In some embodiments, the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord.

In some embodiments, the population of immune cells has comparable (e.g., about 100%, about 100%±5%, about 100%±10%, about 100%±15%, or about 100%±20%) tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.

In some embodiments, the population of immune cells has increased (e.g., at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%) tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.

In another aspect, provided herein is a population of CAR-T cells, wherein (i) the CAR-T cells have a genomic alternation that reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK gene, (ii) at least 70%of the CAR-T cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene, (iii) the CAR-T cells do not have knockout of β2M gene, and (iv) the CAR-T cells have a genomic alteration that reduces or eliminates expression or function of a TCR/CD3 gene.

In another aspect, provided herein is a population of CAR-T cells, wherein (i) the CAR-T cells have one or more genomic alternations that reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, (ii) at least 70%of the CAR-T cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene, (iii) the CAR-T cells do not have knockout of β2M gene, and (iv) the CAR-T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex. In some cases, the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε. In some cases, the CAR-T cells have a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.

Genomic Alteration

Provided herein, in some aspects, are genomic alterations to an immune cell (e.g., a CAR-T cell) or a population of immune cells (CAR-T cells) described herein that reduce or eliminate expression of one or more genes described herein. Genomic alterations can be used to reduce or eliminate expression or function of a protein in an immune cell, a CAR-T cell, a population of CAR-T cells, or a population of immune cells described herein. Genomic alteration refers to any modification or difference of a genome relative to a wild type genome, including nucleotide addition, deletion, substitution, and chemical modification of a nucleotide (e.g., DNA methylation) . Genomic alterations can comprise any change in the DNA sequence that can alter the expression or activity of the protein that it encodes. Genomic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the cell’s genetic material. Such modifications include, for example, disruptions of coding regions and functional fragments thereof for a protein in the referenced species. Additional modifications include, for example, disruptions of non-coding regulatory regions in which the modification alter expression of a gene or operon. Genomic alteration can be achieved by use of any genetic editing systems known to a skilled in the art.

The genomic alteration of the present disclosure can comprise any type of genetic engineering or gene editing technology. In some cases, genomic alterations comprise a mutation, modification or replacement of a DNA. Exemplary genomic alterations comprise insertion, deletion, frameshift mutation, nonsense mutation, gene duplication, missense mutation, substitution, point mutation, silent mutation, or chromosomal inversion of a DNA. One form of genomic alterations can take place through DNA double stranded break (DSB) repair mechanics. Following DSB, two repairing mechanisms that can occur are non-homologous end joining (NHEJ) and homology directed repair (HDR) . NHEJ directly joins the DNA ends. HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point, which causes a programmed change of the genome, including insertion, and replacement of a template DNA. NHEJ can also cause insertions or deletions of base pairs, which cause frameshift mutations.

As used herein, “insertion” refers to a type of mutation comprising the addition of one or more nucleotides into one or more segments of genome. As used herein, “deletion” refers to a type of mutation comprising the removal of one or more nucleotides from one or more segments of genome. As used herein, "substitution" or “replacement” refers to a type of mutation that comprises replacement of one or more nucleotides with one or more different nucleotides.

Gene knockout can refer to a genomic alteration that results in eliminated expression of a gene. A gene knockout can comprise eliminating expression of a gene by introducing a mutation, which comprises nonsense mutation, insertion, deletion, missense mutation, and frameshift mutation. A nonsense mutation or modification can be a mutation in which a sense codon that corresponds to one of the twenty amino acids specified by the genetic code is changed to a chain-terminating codon. A nonsense mutation can generate a truncated, incomplete, and/or nonfunctional protein product. A missense mutation is a point mutation in which a single nucleotide changes results in a codon that codes for a different amino acid. A frameshift mutation can be a genetic mutation caused by indels of a number of nucleotides in a DNA sequence that is not divisible by three.

Suitable nucleases can be used to cause DSB in genomic alteration technologies. Exemplary nucleases are Zinc finger nucleases (ZFNs) , transcription-activator like effector nucleases (TALEN) , meganucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR) system. ZFNs can be a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating DSB in DNA at user-specified locations. TALEN can be made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. TALEN can cut specific sequences of DNA. A CRISPR/Cas9 system can comprise the Cas9 enzyme and a guide RNA. Cas9 can use CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence.

CRISPR/Cas System

In some cases, genomic alteration described herein comprises CRISPR-Cas system. The CRISPR-Cas system comprises Cas enzymes and guide RNAs (gRNAs) that can be part of the bacterial immune system found in nature. A gRNA binds the Cas enzyme and directs it to a genomic DNA target. The Cas enzyme cleaves the DNA target at the targeted site. Knockout of RFX5 and/or TRBC genes of CAR-T cells can reduce the occurrence of adverse reactions such as Graft-versus-host disease (GvHD) and host-versus-graft reactions (HvGR) as described herein.

CRISPR-Cas systems can comprise class 1 and class 2. Class 1 systems can use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems can use a single large Cas protein for the same purpose. Class 1 can be divided into types I, III, and IV. Class 2 can be divided into types II, V, and VI. The 6 system types can be further divided into 19 subtypes. In some cases, the Cas9 of the CRISPR/Cas9 system is replaced by other Cas proteins. In some embodiments, the Cas9 of the CRISPR/Cas9 system is replaced by a Cas protein of class 1 type I, class 1 type III, class 1 type IV, class 2 type II, class 2 type V, or class 2 type VI. In some embodiments, the Cas9 of the CRISPR/Cas9 system is replaced by Cas3, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, Cas12a (Cpf1) , Cas13, Cas14.

In some cases, the Cas protein is isolated from the bacterium Streptococcus pyogenes (SpCas9) , Staphylococcus aureus (SaCas9) , Streptococcus thermophilus (StCas9) , Neisseria meningitidis (NmCas9) , Francisella novicida (FnCas9) , or Campylobacter jejuni (CjCas9) .

In some cases, the Cas protein is a variant derived from the wild type of the Cas proteins. Exemplary Cas variants can be high fidelity Cas9 proteins. In some embodiments, the Cas protein variant have at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acids mutation from the sequence of its wild type. In some embodiments, the Cas variant is fused to another enzyme, for example, activation-induced cytidine deaminase (AID) .

Cas proteins can require a protospacer adjacent motif (PAM) sequence to recognize the target sequence. The PAM sequence can be downstream of the target sequence. The PAM sequence can be 2-6 base pairs in length. PAM sequence for SpCas9 can be NGG. In some embodiments, a Cas variant recognizes an altered PAM sequence. The altered PAM sequences (5 to 3’) can be NGG, NGAN, NGNG, NGAG, NGCG, NNG, TTTN, YTN, or YG (where “Y” is a pyrimidine) .

Guide RNA (gRNA)

In some aspects, provided herein is a gRNA, a RNP complex comprising the gRNA, and a composition comprising the gRNA.

AgRNA can comprise an RNA that functions as a guide for RNA-or DNA-targeting enzymes, with which it forms complexes. A gRNA targets the complementary sequences of a target genome by base pairing. A gRNA can comprise a spacer sequence that is complementary to a corresponding target nucleic acid sequence, referred to as a protospacer. The term “spacer sequence” can include any polynucleotide having sufficient complementarity with a target nucleic acid sequence (i.e., “protospacer” ) to hybridize with the target nucleic acid sequence and direct sequence-specific binding of an effector complex (e.g., CRISPR complex) to the target sequence. In some cases, a spacer sequence is about 20-nt sequence at the 5' end of the gRNA that determines the targeting specificity of CRISPR-Cas9. A guide RNA can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracr sequence) . In some cases, a crRNA comprises a spacer sequence and a tracr mate sequence. In some cases, a crRNA comprises a spacer sequence without a tracr mate sequence. A tracr sequence can form an effector complex with an RNA-guided endonuclease, such as a Cas protein (e.g., Cas9) . In some cases, the tracr mate sequence is hybridized to at least a portion of the tracr sequence. In some cases, the tracr mate sequence and the tracr sequence are connected or linked, for example, by covalent bonds by a linker sequence. The linker sequence can be a sequence of nucleotides which connects the tracr mate sequence and the tracr sequence. In some cases, a gRNA comprises a spacer sequence and a scaffold sequence. A scaffold sequence can be a hairpin structure. In some cases, the scaffold sequence is downstream of the spacer sequence. In some cases, the scaffold sequence comprises a tracr sequence. In some cases, the scaffold sequence comprises a tracr sequence and a tracr mate sequence. In some cases, a gRNA comprises a crRNA, wherein the crRNA comprises a spacer sequence that is connected covalently to a tracr mate sequence. In some cases, a gRNA comprises a spacer sequence, a tracr mate sequence, and a tracr sequence. In some cases, a tracr mate sequence and/or a tracr sequence is downstream of a spacer sequence. Scaffold sequences are known to those of skill in the art and can be obtained from commercial source, such as those described in US Pat. Application No. 20140356958 and US Pat. No. 11261439. A guide RNA can be a two-component species (i.e., separate crRNA and tracr RNA which hybridize together) . A guide RNA can be a one-component species (i.e., a crRNA-tracr RNA fusion, which can be referred to as a single gRNA) .

Adesired target sequence (i.e., protospacer) precedes a protospacer adjacent motif (PAM) . A PAM can be required for a Cas nuclease to cut and can be found 3-4 nucleotides downstream from the cut site. In some cases, after base pairing of the gRNA to the target genome, a Cas9 mediates a double-strand break about 3-nt upstream of PAM. The PAM sequence can be 2-6 base pairs in length. PAM sequence for SpCas9 can be NGG. In some embodiments, a Cas variant recognizes an altered PAM sequence. The altered PAM sequences (5 to 3’) can be NGG, NGAN, NGNG, NGAG, NGCG, NNG, TTTN, YTN, or YG (where “Y” is a pyrimidine) .

In some cases, a gRNA can comprise any one of sequences disclosed in Table 6. In some cases, a gRNA can comprise a sequence having at least 80%sequence identity to any one of sequences disclosed in Table 6.

In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 80%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 81%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 82%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 83%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 84%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 85%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 86%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 87%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 88%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 89%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 90%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 91%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 92%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 93%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 94%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 95%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 96%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 97%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 98%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having at least about 99%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises a sequence having 100%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises at most 3 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises at most 2 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 1-16. In some cases, a gRNA or a polynucleotide encoding the gRNA comprises at most 1 nucleotide mutation relative to the sequence set forth in any one of SEQ ID NOs: 1-16.

In some embodiments, the gRNA comprises a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in any one of SEQ ID NOs: 4-8. In some embodiments, the gRNA comprises a sequence having 0, 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-8. In some embodiments, the gRNA comprises a sequence having at most 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-8. In some cases, the gRNA targets RFX5 gene. In some cases, the gRNA reduces or eliminates expression of RFX5 gene. In some cases, the gRNA reduces or eliminates expression of RFX5 protein. In some cases, the gRNA results in genomic alteration of RFX5 gene.

In some embodiments, the gRNA comprises a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 80%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 90%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 95%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 95%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 96%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 97%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 98%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at least about 99%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having 100%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having 0, 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having at most 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having 1 nucleotide mutation relative to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having 2 nucleotide mutations relative to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having 3 nucleotide mutations relative to the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA comprises a sequence having 4 nucleotide mutations relative to the sequence set forth in SEQ ID NO: 7. In some cases, the gRNA targets RFX5 gene. In some cases, the gRNA reduces or eliminates expression of RFX5 gene. In some cases, the gRNA reduces or eliminates expression of RFX5 protein. In some cases, the gRNA results in genomic alteration of RFX5 gene.

In some embodiments, the gRNA comprises a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in any one of SEQ ID NOs: 9-12. In some embodiments, the gRNA comprises a sequence having 0, 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 9-12. In some embodiments, the gRNA comprises a sequence having at most 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 9-12. In some cases, the gRNA targets RFXANK gene. In some cases, the gRNA reduces or eliminates expression of RFXANK gene. In some cases, the gRNA reduces or eliminates expression of RFXANK protein. In some cases, the gRNA results in genomic alteration of RFXANK gene.

In some embodiments, the gRNA comprises a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in any one of SEQ ID NOs: 13-16. In some embodiments, the gRNA comprises a sequence having 0, 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 13-16. In some embodiments, the gRNA comprises a sequence having at most 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 13-16. In some cases, the gRNA targets RFXAP gene. In some cases, the gRNA reduces or eliminates expression of RFXAP gene. In some cases, the gRNA reduces or eliminates expression of RFXAP protein. In some cases, the gRNA results in genomic alteration of RFXAP gene.

In some embodiments, the gRNA comprises a sequence having 0, 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in SEQ ID NO: 1. In some embodiments, the gRNA comprises a sequence having at most 1, 2, 3, or 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NO: 1. In some embodiments, the gRNA comprises a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 1.

In some cases, efficiency of a genomic alteration (e.g., CRISPR/Cas system) described herein can be measured via any suitable method. For example, TIDE (Tracking of Indels by Decomposition) and targeted deep sequencing. TIDE estimates the spectrum and frequency of small insertions and deletions generated in a pool of cells by genome editing tools such as CRISPR/Cas9 systems. In some cases, knockout efficiency refers to the efficiency of reducing or eliminating the expression of a gene of interest, for example RFX5, RFXANK and/or RFXAP.

Ribonucleoprotein (RNP) complex

In some embodiments, provided herein is a ribonucleoprotein (RNP) complex comprising the gRNA described herein. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having at least about 80%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 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 sequence set forth in any one of SEQ ID NOs: 1-16. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 100%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 4-8. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 9-12. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 13-16. In some embodiments, the RNP complex comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the RNP complex comprises a Cas protein. In some embodiments, the RNP complex comprises a Cas3 protein. In some embodiments, the RNP complex comprises a Cas7 protein. In some embodiments, the RNP complex comprises a Cas8 protein. In some embodiments, the RNP complex comprises a Cas9 protein. In some embodiments, the RNP complex comprises a Cas10 protein. In some embodiments, the RNP complex comprises a Cas11 protein. In some embodiments, the RNP complex comprises a Cas12 protein. In some embodiments, the RNP complex comprises a Cas12a protein. In some embodiments, the RNP complex comprises a Cas13 protein. In some embodiments, the RNP complex comprises a Cas14 protein

Compositions

In some aspects, provided herein is a composition comprising a cell (e.g., the immune cell or an immune cell within the population of immune cells described herein) and a first guide RNA (gRNA) comprising a sequence having at least about 80%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some embodiments, the composition comprises a gRNA comprising a sequence having 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 sequence set forth in any one of SEQ ID NOs: 1-16. In some embodiments, the composition comprises a gRNA comprising a sequence having 100%sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-16. In some embodiments, the composition comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the composition comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 4-8. In some embodiments, the composition comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 9-12. In some embodiments, the composition comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 13-16. In some embodiments, the composition comprises a gRNA comprising a sequence having 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%, at least about 99%, or 100%sequence identity to the sequence set forth in SEQ ID NO: 7. In some cases, the composition further comprises an immune cell that contains the gRNA. In some cases, the immune cell further comprises an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR, e.g., CAR19 described herein) .

Nucleic Acid Molecules, Vectors, and Systems

According to aspects of the present disclosure, provided herein are nucleic acid molecules encoding the gRNA, the CAR, or RNP complex described in the present disclosure.

From the primary amino acid sequence of the polypeptide (s) encoding the CAR and Cas protein provided herein, the person of skill in the art is able to determine suitable nucleotide sequencing (s) that encodes the polypeptide (s) , if desired, one that is codon-optimized (e.g., see Mauro and Chappell. Trends Mol Med. 20 (11) : 604-613, 2014) . From the polynucleotide sequencing of the gRNA provided herein, the person of skill in the art is able to determine suitable polynucleotide sequencing (s) that encodes the gRNA or that is complementary to the gRNA.

The nucleic acid molecule (s) that encode the CAR, gRNA, or RNP complex, such as according to some embodiments of the disclosure, may be, or may be part of, a vector (such as a plasmid vector, cosmid vector or viral vector, or an artificial chromosome) that may comprise other functional regions (elements) such as one or more promoters, one or more origins or replication, one or more selectable marker (s) , and one or more other elements typically found in expression vectors. The cloning and expression of nucleic acids that encode proteins, including CAR and Cas protein, and that encode gRNAs is well established and well within the skill of the person in the art.

In some embodiments, the nucleic acid molecules of the gRNA, CAR, or RNP complex are greater than 80%, such as greater than 90%, greater than 95%, greater than 97%and greater than 99%pure.

In some aspects, provided herein is a vector comprising the nucleic acid sequences encoding a CAR described in the present disclosure. Vector can be a transfer vector, which refers to composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. “Transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. Vector can also include an expression vector, which refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequencing to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the engineered cell or population of cells described herein or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

In some embodiments, the nucleic acid molecule described herein is a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

In some embodiments, the vector is a retroviral vector. A retroviral vector generally refers to an RNA virus that can reverse transcribe a DNA complementary strand in an infected cell, and use this DNA single strand as a template to synthesize a second DNA strand and incorporate it into the cell genome in DNA. The retroviral vector can use enzymes within the engineered cell or population of cells described herein to transcribe and replicate RNA to synthesize proteins, repackage the virus, and release it from the cell to become an infectious virus. The transduction efficiency of the retrovirus can be high, and the transfection rate of the gene can be effectively improved via the retroviral vector.

In some embodiments, the vector is a lentiviral vector. A lentiviral vector refers to the gene therapy vector developed on the basis of HIV-1 (human immunodeficiency type I virus) . The lentiviral vector can infect both dividing cells and non-dividing cells. It can effectively infect almost all mammalian cells including neuron cells, liver cells, etc., with high infection efficiency. Lentiviruses can efficiently integrate foreign genes into the host chromosomes to achieve persistent expression. In some embodiments, the nucleic acid molecules and/or vector of the present disclosure is introduced into a producer cell, such as a stable, 293T-derived producer cell line. In some cases, the producer cells generate lentiviral particles comprising nucleic acid molecules encoding the CARs. In some cases, the lentiviral particles can be introduced to the engineered cell or population of cells described herein.

In some embodiments, the vector is a transposon plasmid. A transposon plasmid generally refers to the basic unit existing on chromosomal DNA and capable of autonomous replication and displacement. The transposon plasmid can "jump" from one position of the genome to another through a series of processes such as cutting and reintegration.

In some embodiments, the vector is an expression vector. In some embodiments, the expression vector comprises a nucleic acid sequence encoding a CAR (e.g., a CAR19) . In some embodiments, the expression vector comprises a nucleic acid sequence encoding a Cas protein that forms a RNP complex with a gRNA described herein.

In some embodiments, the nucleic acid molecules and/or vector of the present disclosure is introduced into the engineered cell (e.g., engineered T cells) or population of cells described herein. For eukaryotic cells, for example, suitable techniques include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. In some cases, introducing nucleic acid in the engineered cell or population of cells described herein, in particular a eukaryotic cell, uses a viral or a plasmid-based system. In some cases, the plasmid system is maintained episomally. In other cases, the plasmid system is incorporated into the engineered cell or population of cells described herein or into an artificial chromosome. In a particular embodiment, the incorporation is by random integration of one or more copies at single or multiple loci. In some embodiments, the incorporation is by targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include, for example, calcium chloride transformation, electroporation and transfection using bacteriophage.

In some embodiments, the nucleic acid of the present disclosure is integrated into the genome (e.g., chromosome) of the engineered cell (e.g., engineered T cells) or population of cells described herein. In a particular embodiment, integration is promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques. In some embodiments, the nucleic acid sequence encoding a CAR is present in a genome of the cell.

According to aspects of the present disclosure, provided herein are systems relating to the nucleic acid molecules described herein. A system can comprise the nucleic acid molecule or the vector described herein. In some embodiments, a system comprises one nucleic acid molecule or one plasmid, which encodes for a CAR (e.g., CAR19) .

Methods and Uses

Provided herein are a methods and uses related to the immune cell, the population of immune cells, the gRNA, the RNP complex comprising the gRNAs, the compositions, and the pharmaceutical compositions as described herein.

Engineering an Immune Cell

In aspects, provided herein is a method of engineering an immune cell, the method comprising introducing gRNAs described herein into the immune cell or contacting the immune cell with the RNP complex described herein.

In some cases, the method comprises introducing into the immune cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-8; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-8, thereby resulting in genomic alteration of RFX5 gene in the immune cell. In some cases, the method comprises introducing into the immune cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 9-12; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 9-12, thereby resulting in genomic alteration of RFXANK gene in the immune cell. In some cases, the method comprises introducing into the immune cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 13-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 13-16, thereby resulting in genomic alteration of RFXAP gene in the immune cell. In some cases, the method comprises electroporating the cell with reagents comprising the gRNA or the RNP complex. In some cases, the method further comprises contacting the cell with a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) . In some cases, the nucleic acid molecule is a vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some cases, the cell is an immune cell, optionally a CD4+ T cell or a CD8+ T cell. In some cases, the cell is isolated from cord blood of a human or is a progeny of a cell isolated from cord blood of a human.

In some cases, to obtain the immune cell or population of immune cells described herein, a cell or a group of cells (e.g., T cells obtained from cord blood) is treated so as to cause or allow genomic alteration (s) by an RNP complex described herein, e.g., by culturing engineered cell or population of cells described herein under conditions to allow introduction of an RNP complex described herein and allow the genomic alteration functioning achieved by the RNP complex within the engineered cell or population of cells described herein. In some cases, to obtain the immune cell or population of immune cells described herein, the cell or group of cell (e.g., T cells obtained from cord blood) is treated so as to allow expression of a CAR described herein, e.g., by culturing immune cell or population of cells described herein under conditions to allow expression of the CAR (e.g., CAR19) . In some embodiments, the purification of the expressed product is achieved by methods known to one of skill in the art. For example, the expression vector or reagents comprising gRNA described herein can be transferred into the engineered cell or population of cells described herein by physical, chemical, or biological means. A nucleic acid with the spacer sequences (e.g., described in Table 8) can be separated by the restriction enzyme cut sites and can be introduced into a plasmid. Each restriction enzyme cut site can be cut and a nucleic acid sequence encoding a guide RNA scaffold sequence can be inserted therein. Promoter sequences and other regulatory sequences required for expression can also be included so that a nucleic acid is created that encodes one or more guide RNAs can be expressed. In addition, the guide RNA scaffold sequence can be modified or can include insertion sequences that alter or provide certain Cas9 functionality.

Physical methods for introducing a polynucleotide (e.g., gRNA described herein) or reagents comprising the polynucleotide (e.g., RNP complex described herein) into the engineered cell or population of cells described herein include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acid molecules are well-known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY) . In some cases, a method for the introduction of a polynucleotide (e.g., gRNA described herein) or reagents comprising the polynucleotide (e.g., RNP complex described herein) into the engineered cell or population of cells described herein is calcium phosphate transfection.

Biological methods for introducing a polynucleotide (e.g., encoding a CAR such as CAR19) into the engineered cell or population of cells described herein include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide (e.g., gRNA) or reagents comprising the polynucleotide (e.g., RNP complex described herein) into the engineered cell or population of cells described herein include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) . Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into the engineered cell or population of cells described herein (in vitro, ex vivo or in vivo) . In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid molecules associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ( “DMPC” ) can be obtained from Sigma, St. Louis, Mo. ; dicetyl phosphate ( “DCP” ) can be obtained from K & K Laboratories (Plainview, N.Y. ) ; cholesterol ( “Choi” ) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ( “DMPG” ) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala. ) . Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 ℃. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10) . However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce nucleic acid molecules, vectors, or systems described herein into the engineered cell or population of cells described herein, in order to confirm the presence of the recombinant DNA sequence in the engineered cell or population of cells described herein, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.

In some cases, the method further comprises contacting the group of cells with a nucleic acid molecule comprising an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) , prior to contacting with the reagents. In some cases, the method further comprises contacting the group of cells with a nucleic acid molecule comprising an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) , subsequent to contacting with the reagents. In some cases, subsequent to contacting the cell or the group of cells with the reagents, the methods further comprise a selection step. In some cases, cells are sorted using CD3 MicroBeads. In some cases, the selection step comprises contacting the cell or the group of cells with CD3 MicroBeads to obtain cells with a TCR-ratio of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%. In some cases, the selection step comprises contacting the cell or the group of cells with CD3 MicroBeads to obtain cells with a TCR-ratio of about 99.9%.

Reducing Expression of MHC-I Molecules and Maintaining Tolerance to NK Cells

In another aspect, provided herein is a method of reducing expression of HLA-A, HLA-B, or HLA-C gene while maintaining tolerance of an immune cell to host NK cells, comprising genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein in the immune cell. Also provided herein is a method of reducing expression of HLA-A, HLA-B, or HLA-C gene while maintaining tolerance of an immune cell to host NK cells, comprising genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK genes in the immune cell. In some embodiments, the method does not comprise knocking out β2M gene in the immune cell. In some embodiments, the method does not comprise knocking down β2M gene in the immune cell. In some embodiments, the method does not comprise knocking out or knocking down β2M gene in the immune cell.

In some embodiments, the method does not comprise knocking out a NK activating receptor ligand gene in the immune cell. In some embodiments, the method does not comprise knocking down a NK activating receptor ligand gene in the immune cell. In some embodiments, the method does not comprise knocking out or knocking down a NK activating receptor ligand gene in the immune cell. Exemplary NK activating receptor ligand genes include MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60, MULT1, B7-H6, BAG6, PfEMP1, HSPGS, AICL, CD112, CD155, CD48, CD58, CD59, ICAM1, ICAM2, ICAM3, STAT1, JAK1, IFNGR2, JAK2, or IFNGR1.

In some embodiments, the method does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cell. Exemplary NK inhibitory molecules include antibody or an antigen binding fragment thereof targeting an NK-inhibiting receptor, wherein the NK-inhibiting receptor is selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM 1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4, and KLRG1. In some embodiments, the NK inhibitory molecule comprises a transmembrane domain, and a costimulatory domain.

In some embodiments, the immune cell is a lymphocyte or a myeloid cell. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell. In some embodiments, the lymphocyte is a CD8+ T cell or a CD4+ T cell. In some embodiments, the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) . In some embodiments, the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a TCR/CD3 complex component. The TCR/CD3 complex component can be encoded by TRAC, TRBC, CD247, CD3G, CD3D, CD3E genes, or any combination thereof.

In some embodiments, the immune cell is a myeloid cell. The term “myeloid cell” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from myeloid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, granulocytes (i.e., mast cells, neutrophils, eosinophils and basophils) , monocytes, macrophages, and dendritic cells.

In some embodiments, the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord. In some embodiments, the immune cells are derived from an autologous donor or an allogenic donor. In some embodiments, the immune cells are derived from an autologous donor. In some embodiments, the immune cells are derived from an allogenic donor.

In some embodiments, the immune cell is from peripheral blood, bone marrow, placenta, or umbilical cord. In some embodiments, the immune cells are from an autologous donor or an allogenic donor. In some embodiments, the immune cells are from an autologous donor. In some embodiments, the immune cells are from an allogenic donor.

Genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK proteins in the immune cell can be achieved by use of any gene editing systems known in the art. Genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK genes in the immune cell can be achieved by use of any gene editing systems known in the art. In some embodiments, the gene editing system is a CRISPR system.

Accordingly, in another aspect, provided herein is a guide RNA (gRNA) comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to the sequence selected from SEQ ID NOs: 4-16. In one aspect, provided herein is a guide RNA (gRNA) or a polynucleotide encoding the guide RNA, the guide RNA comprising a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-16.

Also provided herein is a ribonucleoprotein (RNP) complex comprising the gRNA described herein and a Cas protein.

Also provided herein is a composition comprising the gRNA described herein. In some cases, the composition comprises an immune cell that contains the gRNA. In some cases, the immune cell further comprises an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) .

Also provided is a method of reducing or eliminating expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein in an immune cell, comprising introducing any of the gRNA disclosed herein to the immune cell.

Also provided is a method of reducing or eliminating expression and/or function of one or more of RFX5, RFXAP, and RFXANK gene in an immune cell, comprising introducing any of the gRNA disclosed herein to the immune cell.

Therapeutic Uses

Also provided herein are methods for treating a subject in need thereof comprising administering to the subject an effective amount of any of the immune cell or cell population provided herein. Also provided herein is a metho comprising administering the population of immune cells described herein, the population of CAR-T cells described herein, the immune cell described herein, or the CAR-T cell described herein, to a subject in need thereof. Also provided herein are methods for inhibiting tumor growth or cancer progression in a subject in need thereof. In some cases, the methods comprise administering to the subject a pharmaceutical composition described herein. In some embodiments of the methods disclosed herein, the immune cell or cell population is administered systemically, intranasally, intrapleurally, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the subject is human. In some embodiments, the subject is human. In some embodiments, a therapeutically effective amount of the engineered cell or the population of cell provided herein is administered.

Also provided herein is use of the immune cell, the population of immune cells, the CAR-T cell, or the population of CAR-T cells of described herein in an adoptive cell therapy.

The subject can have a certain disease or a condition in need of treatment provided by the present disclosure. The disease or condition can include, e.g., reducing or ameliorating, a hyperproliferative condition or disorder, for example a cancer. The disease or condition can include but not limited to, solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term “cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.

Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon) , genitourinary tract (e.g., renal, urothelial cells) , prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

Exemplary cancers whose growth can be inhibited include cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include melanoma (e.g., metastatic malignant melanoma) , renal cancer (e.g., clear cell carcinoma) , prostate cancer (e.g., hormone refractory prostate adenocarcinoma) , breast cancer, colon cancer and lung cancer (e.g., non-small cell lung cancer) . Additionally, refractory or recurrent malignancies can be treated using the molecules described herein.

In some embodiments, the method treats cancer in a subject. In some embodiments, the method treats a solid tumor. In some embodiments, the cancer comprises leukemia. In some embodiments, the cancer comprises melanoma. In some embodiments, the cancer comprises lymphoma. In some embodiments, the cancer comprises adrenal gland cancer, bladder cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, fallopian tube cancer, gastrointestinal cancer, glioma, glioblastoma, head and neck cancer, hematopoietic malignancy, leukemia, liver cancer, lung cancer, lymphoma, myeloma, nasal cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, squamous cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, or any combination thereof.

In some cases, the method provided herein treats acute lymphoblastic leukemia (ALL) . ALL can comprise the malignant proliferation of lymphoid progenitor or precursor cells comprising small to medium-sized blast cells involving bone marrow and blood. ALL can be characterized by an excess of malignant lymphoblasts. The majority of ALL malignancies can be of B-cell origin, named acute B-lymphoblastic leukemia (B-ALL) . In some cases, CD19 is the earliest of the B-lineage-restricted antigens and B-ALL cancer cells express CD19. The engineered cell or group of cells expressing CAR19 can recognize and target CD19 expressing cells, including B-ALL cancer cells and normal B cells. After specific binding with CD19 expressing cells, the engineered cell or group of cells (e.g., allogeneic CAR-T cells with one or more genomic alterations) can specifically kill target cells. In some cases, the method provided herein treats acute CD19 positive B lymphoid malignancies in adult. In some cases, the method provided herein treats acute CD19 positive B lymphoid malignancies in pediatric populations.

For treatment, the amount of the immune cell or immune cell population provided herein administered is an amount effective in producing the desired effect, for example, treatment or amelioration of the effects and/or symptoms of tumors in a subject in need thereof.

An effective amount can be provided in one or a series of administrations of the immune cell or immune cell population provided herein. An effective amount can be provided in a bolus or by continuous perfusion.

For adoptive immunotherapy using the immune cell or immune cell population (e.g., CAR-T cells) provided herein, while cell doses in the range of about 10⁶ to about 10¹⁰ are typically infused, lower doses of the immune cells can be administered, e.g., about 10⁴ to about 10⁸. Methods for administering cells for adoptive cell therapies, including, for example, donor lymphocyte infusion and CAR-T cell therapies, and regimens for administration are known in the art and can be employed for administration of the engineered immune cells provided herein.

The immune cell or immune cell population of the presently disclosed subject matter can be administered by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, the immune cell or the population of immune cells and the compositions comprising the same are intravenously administered to the subject in need. A suitable pharmaceutically acceptable carrier for the cells for injection can include any isotonic carrier such as, for example, normal saline (about 0.90%w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water) , NORMOSOL R electrolyte solution (Abbott, Chicago, Ill. ) , PLASMA-LYTE A (Baxter, Deerfield, Ill. ) , about 5%dextrose in water, or Ringer's lactate. In some embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

For therapeutic applications, a pharmaceutical composition comprising the immune cell or immune cell population of the present disclosure, is administered to the subject. In some embodiments, the immune cell or immune cell population of the present disclosure is administered one, two, three, four, or five times per day. In some embodiments, the immune cell or immune cell population of the present disclosure is administered more than five times per day. Additionally or alternatively, in some embodiments, the immune cell or immune cell population of the present disclosure is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the immune cell or immune cell population of the present disclosure is administered weekly, bi-weekly, tri-weekly, or monthly. In some embodiments, the immune cell or immune cell population of the present disclosure is administered for a period of one, two, three, four, or five weeks. In some embodiments, the engineered immune cells are administered for six weeks or more. In some embodiments, the engineered immune cells are administered for twelve weeks or more. In some embodiments, the engineered immune cells are administered for a period of less than one year. In some embodiments, the engineered immune cells are administered for a period of more than one year. In some embodiments, the engineered immune cells are administered throughout the subject’s life.

In some embodiments of the methods of the present disclosure, the immune cell or immune cell population of the present disclosure is administered daily for 1 week or more. In some embodiments of the methods of the present disclosure, the immune cell or immune cell population of the present disclosure is administered daily for 2 weeks or more. In some embodiments of the methods of the present disclosure, the immune cell or immune cell population of the present disclosure is administered daily for 3 weeks or more. In some embodiments of the methods of the present disclosure, the immune cell or immune cell population of the present disclosure is administered daily for 4 weeks or more. In some embodiments of the methods of the present disclosure, the immune cell or immune cell population of the present disclosure is administered daily for 6 weeks or more. In some embodiments of the methods of the present disclosure, the immune cell or immune cell population of the present disclosure is administered daily for 12 weeks or more. In some embodiments, the engineered immune cells are administered throughout the subject’s life.

The presently disclosed subject matter provides various methods of using the immune cell or immune cell population (e.g., a CAR-T cell) provided herein. Additionally or alternatively, in some embodiments, the immune cell or immune cell population provided herein further expresses one or more cell-surface ligands that bind to additional targets. Examples of such additional targets include, but are not limited to GRAMD1A, KCNK3, RAI2, NPL, STC1, TOM1, F3, SLC6A8, SLC22A4, SERINC3, DDIT4L, LY96, NFASC, IFNGR1, DNER, SLC22A1, ITGB3, LRP10, ICAM1, ULBP2, SLC22A15, APLPl, ABTB2, AFF1, AGPAT2, AGTRAP, AKAP6, BFSP1, BHLHE40, CARD6, CCDC69, CCDC71L, FAM219A, FAM219B, FAM43A, FAM8A1, FOLR3, GSAP, GYS1, HECW2, HIF1A, INHBA, MAP3K8, MT-ND5, MT-ND6, and PRICKLE2. Other examples of such additional targets include, but are not limited to LRP12, SLC6A8, ITGB3, LRP10, BTN2A2, ICAM1, ABCAl, SLC22A23, TMEM63B, SLC37A1, SLC22A4, ENPP4, VNN1, SERINC3, ITGA11, SERINC2, ULBP2, SLC22A15, APLPl, DPP4, ABC A3, TPCN1, ABTB2, AFF1, AGPAT2, AGTRAP, AHNAK2, AK4, AKAP6, ALS2CL, AMPD3, ANKRD1, ANKRD29, ANKRD42, AOX1, ARHGEF37, ARRDC4, ATP6V1H, BFSP1, BHLHE40, BHLHE41, BTG2, C3, CARD6, CASP4, CCDC69, CCDC71L, CDKN1A, CHST15, COQIOB, CPPED1, CTSB, CYB5R1, CYBA, CYFIP2, CYP26B1, DDIT4L, DIRC3, DNAJB9, DTX4, DYNLT3, ELL2, ELOVL7, EML1, FADS3, FAM210B, FAM219A, FAM219B, FAM43A, FAM8A1, FILIP 1L, FOLR3, FOXOl, GFPT2, GM2A, GPX3, GRAMD1A, GRBIO, GSAP, GYS1, HECW2, HIF1A, HIST2H2BE, IDS, IGFN1, INHBA, JUN, KCNJ15, KCNK3, KDM6B, KIAA1217, KLHL21, LCP1, LINC00862, LY96, LYPLALl, LZTS3, MAP1LC3B, MAP3K10, MAP3K8, MAP7, MAPRE3, MAST3, MOAPl, MSC, MT-ND3, MT-ND5, MT-ND6, MXD1, MYOID, NABPl, NOV, NPL, OGFRLl, P4HA2, PGM2L1, PHYH, PLA2G15, PLA2G4C, PLD1, PLEKHG5, PLOD2, PPARGCIA, PPP2R5B, PRICKLE2, PSAP, RAB29, RAB36, RAB6B, RAG1, RAI2, RETSAT, RIOK3, RNF11, RNF14, RSPH3, RUSC2, SAT1, SCG5, SEL1L3, SERPINI1, SESN2, SIAE, SOD2, SPATA18, SPTBN2, SRPX2, ST20-AS1, STC1, STK38L, STON2, SUSD6, TAF13, TAPI, TBC1D2, TFEC, TNFAIP3, TNFAIP8L3, TOM1, TPRG1L, TSKU, TTC9, TXNIP, UBA6-AS1, VPS 18, WDR78, ZFHX2, and ZNFX1.

The presently disclosed subject matter also provides methods of increasing or lengthening survival of a subject with cancer. In one non-limiting example, the method of increasing or lengthening survival of a subject with cancer comprises administering an effective amount of the presently disclosed immune cell or immune cell population to the subject, thereby increasing or lengthening survival of the subject. The presently disclosed subject matter further provides methods for treating cancer in a subject, comprising administering the presently disclosed immune cell or immune cell population to the subject.

The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.

Further modification can be introduced to the immune cell or immune cell population to avert or minimize the risks of immunological complications, e.g., graft-versus-host disease (GvHD) , host-versus-graft reaction (HvGR) , or in the case of T cells, “malignant T-cell transformation. ” Modification of the immune cell or immune cell population can include engineering a suicide gene into the immune cell or immune cell population. Suitable suicide genes include, but are not limited to, Herpes simplex virus thymidine kinase (hsv-tk) , inducible Caspase 9 Suicide gene (iCasp-9) , and a truncated human epidermal growth factor receptor (EGFRt) polypeptide. The incorporation of a suicide gene into a presently disclosed immune cell or immune cell population gives an added level of safety with the ability to eliminate the majority of the immune cell or immune cell population within a very short time period. A presently disclosed immune cell or immune cell population incorporated with a suicide gene can be pre-emptively eliminated at a given time point post cell infusion, or eradicated at the earliest signs of toxicity.

In one aspect, the present disclosure provides methods for treating or ameliorating cancer in a subject that has received or is receiving radiation therapy or chemoradiation therapy comprising administering to the subject a therapeutically effective amount of any immune cell or immune cell population described herein. In another aspect, the present disclosure provides a method for improving the efficacy of adoptive cell therapy in a subject diagnosed with cancer comprising administering to the subject an effective dose of radiation therapy or chemoradiation therapy and a therapeutically effective amount of any of the immune cell or immune cell population described herein. In some embodiments, the subject is diagnosed as having, suspected as having, or at risk of having cancer.

In therapeutic applications, pharmaceutical compositions or medicaments comprising immune cell or immune cell population of the present disclosure, are administered to a subject suspected of, or already suffering from cancer, in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease, including its complications and intermediate pathological phenotypes in development of the disease or condition.

Subjects suffering from cancer can be identified by any or a combination of diagnostic or prognostic assays known in the art.

Methods for treating a subject in need thereof may further comprise sequentially, separately, or simultaneously administering to the subject at least one additional therapy, e.g., chemotherapy, radiation therapy, or another immunotherapy.

In any case, the multiple therapeutic agents can be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents can be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills) . One of the therapeutic agents can be given in multiple doses, or both can be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.

Administration

The immune cell or cell population of the presently disclosed subject matter can be provided systemically or directly to a subject in need thereof. In certain embodiments, the immune cell or cell population is directly injected into an organ of interest. Additionally or alternatively, the immune cell or cell population is provided indirectly to the organ of interest, for example, by administration into the circulatory system or into the tissue of interest. Expansion and differentiation agents can be provided prior to, during or after administration of cells and compositions to increase production of the immune cell or cell population (e.g., T cell or T cell population) in vitro or in vivo.

The immune cell or cell population of the presently disclosed subject matter can be administered in any physiologically acceptable vehicle, systemically or regionally, normally intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus) . In certain embodiments, at least 1×10⁵ cells can be administered, eventually reaching 1×10¹⁰ or more. In certain embodiments, at least 1×10⁶ cells can be administered. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage) . The engineered immune cells can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g., IL-2, IL-3, IL 6, IL-11, IL-7, IL-12, IL-15, IL-21, as well as the other interleukins, the colony stimulating factors, such as G-, M-and GM-CSF, interferons, e.g., g-interferon.

In certain embodiments, compositions of the presently disclosed subject matter comprise pharmaceutical compositions comprising the immune cell or cell population of the presently disclosed subject matter with a pharmaceutically acceptable carrier. Administration can be autologous or non-autologous.

PHARMACEUTICAL COMPOSITIONS

The immune cell or cell population of the presently disclosed subject matter can be conveniently provided as a pharmaceutical composition, for example, sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) ; and preservatives. Compositions of the present disclosure are in one embodiment formulated for intravenous administration. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose) , pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON' S PHARMACEUTICAL SCIENCE” , 17th edition, 1985, incorporated herein by reference, can be consulted to prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the engineered immune cells of the presently disclosed subject matter.

The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter can be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form) .

In some embodiments, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL) , p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In some embodiments, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the engineered immune cells as described in the presently disclosed subject matter. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation) , from this disclosure and the documents cited herein.

One consideration concerning the immune cell or cell population of the presently disclosed subject matter is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 10² to about 10¹², from about 10³ to about 10¹¹, from about 10⁴ to about 10¹⁰, from about 10⁵ to about 10⁹, or from about 10⁶ to about 10⁸ engineered immune cells of the presently disclosed subject matter are administered to a subject. More effective cells can be administered in even smaller numbers. In some embodiments, at least about 1×10⁸, about 2×10⁸, about 3×10⁸, about 4×10⁸, about 5×10⁸, about 1×10⁹, about 5×10⁹, about 1×10¹⁰, about 5×10¹⁰, about 1×10¹¹, about 5×10¹¹, about 1×10¹² or more engineered immune cells of the presently disclosed subject matter are administered to a human subject. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Generally, engineered immune cells are administered at doses that are nontoxic or tolerable to the patient.

The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions to be administered in methods of the presently disclosed subject matter. Typically, any additives (in addition to the active cell (s) and/or agent (s) ) are present in an amount of from about 0.001%to about 50%by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt %to about 5 wt%, from about 0.0001 wt%to about 1 wt%, from about 0.0001 wt%to about 0.05 wt%, from about 0.001 wt%to about 20 wt%, from about 0.01 wt%to about 10 wt %, or from about 0.05 wt%to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition (s) , concentration of components therein and timing of administering the composition (s) , which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And the time for sequential administrations can be ascertained without undue experimentation.

Kits

In one aspect, the kits of the present disclosure comprise a therapeutic composition including any of the immune cell or immune cell population disclosed herein in unit dosage form, and/or vectors comprising any of the nucleic acids disclosed herein. In some embodiments, the kit comprises a sterile container which contains therapeutic compositions including the immune cell or immune cell population disclosed herein; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

In some embodiments of the kits, the immune cell or immune cell population of the present disclosure can be provided together with instructions for administering the immune cell or immune cell population to a subject. In some embodiments, the subject is diagnosed with or suffers from cancer. Additionally or alternatively, in some embodiments, the subject suffering from cancer has received or is receiving radiation therapy or chemoradiation therapy.

In certain embodiments of the kits, the vectors comprising any of the gRNAs disclosed herein can be provided together with instructions for using immune cell or immune cell population transduced with said vectors to treat or mitigate any disease or condition described herein.

The instructions will generally include information about the use of the composition for the treatment of any disease or condition described herein. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment of any disease or condition described herein or symptoms thereof; precautions; warnings; indications; counter indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions can be printed directly on the container (when present) , or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The immune cell or immune cell population of the present disclosure can be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation (e.g., Kivitz et al., Clin. Ther. 28: 1619-29 (2006) ) .

Adevice capable of delivering the kit components through an administrative route can be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices.

The kit components can be packaged together or separated into two or more containers. In some embodiments, the containers can be vials that contain sterile, lyophilized formulations of engineered immune cell compositions of the present disclosure that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents. Other containers that can be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components can be packaged and maintained sterilely within the containers.

EXAMPLES

The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

Example 1. Methods and Materials

Construction of CRISPR/Cas9 system

Agents and Instruments:

(1) . Alt-R CRISPR-Cas9 Nuclease V3 (500μg, cat: 1081061, Integrated DNA Technologies (IDT) ) ;

(2) . Alt-R CRISPR-Cas9 tracrRNA (100nmol, cat: 1072534, IDT) ;

(3) . Alt-R CRISPR-Cas9 Electroporation Enhancer (10 nmol, cat: 1075916, IDT) ;

(4) . Nuclease-Free Duplex Buffer (IDT) ;

(5) . Alt-R CRISPR-Cas9 crRNA (10nmol, customized, IDT) ;

(6) . Opti-MEM (500ml, cat: 11058021, Thermo) ;

(7) . Flow cytometer (NovoCyte Penteon, Agilent) ;

(8) . electroporation instrument (CTX-1500ALE, Celetrix) ; and

(9) . electroporation cup (1207, Celetrix) .

Experiment Procedure Step 1-RNA preparation:

(1) Design and synthesize crRNA on the IDT official website;

(2) Dissolve each RNA with Nuclease-Free Duplex Buffer, the dissolved volume and concentration are shown in Table 1;

Table 1

Note: Store the stock solution at -20℃.

(3) Mix crRNA and tracrRNA in equimolar concentrations in a sterile centrifuge tube, see Table 2 for the configuration method;

Table 2

(4) Heat the sterile centrifuge tube at 95℃ for 5 minutes;

(5) Take out the centrifuge tube and let it stand at room temperature until its temperature is between 20-25℃;

Note: gRNA is stable at -20℃ for at least 6 months without loss of activity.

Experiment Procedure Step 2-RNP complex formation:

(1) Take out the Alt-R Cas9 enzyme, centrifuge at 300g for 5min, and mix by pipetting before use;

(2) Configure RNP, see Table 3 for each component and the amount added;

Table 3. RNP configuration

(3) Let the RNP stand at room temperature for 5 min.

Note: RNP complexes can be stored at 4 ℃ for 4 weeks, or at -80 ℃ for 6 months.

Experiment Procedure Step 3-transfecting cells with RNP:

(1) Centrifuge the cells to be edited at 300g for 5min, and resuspend them in Opti-MEM at a density of 5-6×10⁶ cells/ml;

(2) Configure the electroporation system, see Table 4 for the amount of RNP and cell suspension added;

Table 4. Electroporation system configuration

(3) Pipette and mix the configured electroporation system and transfer it to the electroporation cup, and set the parameters of the electroporation instrument:

Voltage: 500V, pulse time: 20ms, electroporation;

(4) After electroporation, aspirate the cells from the electroporation cup and place them in 1ml 1640 medium (containing 10%FBS) ;

(5) Transfer the cells to a 37℃, 5%CO₂ incubator, let stand for 30 minutes, then check the cell density and adjust it to 4×10⁵ cells/ml

The gRNAs used in the experiments described in Examples 1-5 are summarized in Table 6 below.

Table 6. Exemplary spacer sequences of gRNA and their corresponding PAM sequences

Construction of CAR

Experimental procedure:

The following protein sequences were synthesized and cloned sequentially into the pK14 vector: CD8α signal peptide (SEQ ID NO: 18) , anti-CD19 scFv (SEQ ID NO: 20) , CD8α hinge region (SEQ ID NO: 22) , CD8α transmembrane region (SEQ ID NO: 24) , 4-1BB co-stimulatory domain (SEQ ID NO: 26) , CD3ζ intracellular signaling domain (SEQ ID NO: 28) , and the correct insertion of the target sequence was confirmed by sequencing. After adding 5ml of Optim-MEM to a sterile 15ml centrifuge tube, add the virus packaging vector and virus envelope vector according to the ratio of pK14-CD19: pLP1: pLP2: pLP-VSVG=5: 4: 3: 1, and then add 800 μL PEI transfection reagent, mix immediately, incubate at room temperature for 15 min, and then add the plasmid/vector/transfection reagent complex dropwise into the culture flask of 293T cells. Virus supernatants were collected at 24h and 48h, combined, and ultracentrifuged (25000g, 4℃, 3h) to obtain concentrated lentivirus.

Information of the CAR

Signal peptide (CD8α) :

Nucleic acid sequence:

gccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg (SEQ ID NO: 17) ;

Amino acid sequence: ALPVTALLLPLALLLHAARP (SEQ ID NO: 18) .

scFv (CD19) :

Nucleic acid sequence:

Amino acid sequence:

Hinge (CD8α) :

Nucleic acid sequence:

Amino acid sequence:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 22) .

Transmembrane domain (CD8α) :

Nucleic acid sequence:

Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc (SEQ ID NO: 23) ;

Amino acid sequence: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 24) .

Costimulatory domain (4-1BB) :

Nucleic acid sequence:

Amino acid sequence:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 26) .

Intracellular signal domain (CD3ζ)

Nucleic acid sequence:

Amino acid sequence:

Complete sequence of CAR (CD8α Leader+Anti CD19-scFv+CD8α Hinge+CD8α Transmembrane+4-1BB+CD3ζ)

Nucleic acid sequence:

Amino acid sequence:

Example 2. Tolerance of RFX5-KO T cells to NK cells compared with β2M-KO T cells

This example illustrates that RFX5-KO T cells have improved tolerance to NK cells, as compared to corresponding β2M-KO T cells.

Preparation, culture, and cryopreservation of immune tolerance RFX5-KO T cells

1. Day 0, Resuscitation of Cord Blood or Peripheral Blood

Remove the cord blood or peripheral blood from the liquid nitrogen and shake it quickly in a 37℃ water bath until it completely melts. After the surface of the umbilical cord blood or peripheral blood bag was disinfected, it was transferred to a biological safety cabinet. Transfer the umbilical cord blood or peripheral blood from the blood bag to a 50mL centrifuge tube, add 1640 medium (10 times the volume of the cord blood or peripheral blood) , mix well and centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 20mL of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to resuspend the cells, add T cell activation antibody, and transfer the cells to a 37℃, 5%CO₂ incubator to stand for 48h.

2. Day 2, Purification of T Cells

2.1 CD4+ T cells

After 48h, the cells in the culture flask were mixed and transferred to a 50mL centrifuge tube, centrifuged at 300g for 5min. After centrifugation, discard the supernatant, add 20mL MACS buffer to resuspend the cells, and centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 40 μL MACS CD4 immunomagnetic beads, mix well and incubate in the dark for 15 minutes, add 20 mL MACS buffer, centrifuge at 300g for 5 minutes. Assemble the separation column of MACS and wipe the surface with alcohol to disinfect, and rinse the separation column with 3mL MACS buffer. After centrifugation, discard the supernatant, add 1mL MACS buffer to resuspend the cells, add the cells to the washed MACS separation column, and then rinse the separation column with MACS buffer twice, 3mL/time. After the MACS buffer has run out, get 10mL of cells in the lower layer, replace the centrifuge tube, remove the MACS sorting column, add 5mL of MACS buffer, use the booster to punch out the MACS buffer, and get 5mL of the upper layer cells. Mix the upper and lower layer cells separately, and sample 20 μL for flow cytometry staining and counting. The upper layer cells are purified CD4+ T cells.

2.2 CD8+ T cells

Centrifuge 10 mL of the lower layer cells after CD4 sorting at 300 g for 5 min. After centrifugation, discard the supernatant, add 40 μL MACS CD8 immunomagnetic beads, mix well and incubate in the dark for 15 minutes, add 10 mL MACS buffer, centrifuge at 300 g for 5 minutes. Assemble the separation column of MACS and wipe the surface with alcohol to disinfect, and rinse the separation column with 3mL MACS buffer. After centrifugation, discard the supernatant, add 1mL MACS buffer to resuspend the cells, add the cells to the washed MACS separation column, and then rinse the separation column with MACS buffer twice, 3mL/time. After the MACS buffer has run out, get 10mL of cells in the lower layer, replace the centrifuge tube, remove the MACS sorting column, add 5mL of MACS buffer, use the booster to punch out the MACS buffer, and get 5mL of the upper layer of cells. Mix the upper and lower layer cells separately, and sample 20 μL for flow cytometry staining and counting. The upper layer of cells is purified CD8+ T cells.

3. On Day 2, Gene Editing of T Cells

3.1 Configure gRNA

Add 1 μL each of crRNA (including β2M-sgRNA, RFX5-GH-sgRNA) and tracrRNA to 98 μL Nuclease-Free Duplex Buffer, the total volume is 100 μL. Heat the gRNA at 95℃ for 5 minutes, take it out and let it stand at room temperature until its temperature returns to 20-25℃. Remarks: Each gRNA is configured separately.

3.2 Configure RNP

Add 1.5 μL of gRNA (including β2M, RFX5-GH-sgRNA) , 1.5 μL of Cas9, and 0.6 μL of Enhancer to 21.4 μL of Opti-MEM, with a total volume of 25 μL. Let the RNP stand at room temperature for 5 minutes. Remarks: Each RNP is configured separately.

3.3 Configure the electroporation system and electroporation

Centrifuge the purified CD4+ and CD8+ T cells separately, 300g, 5min, resuspend in Opti-MEM, the resuspension density is 5～6×10⁶ cells/mL, take 25μL CD4+ or CD8+T cell suspension and mix with 25μL RNP, transferred to the electroporation cup, set the electroporation parameters, voltage: 500V, pulse time: 20ms, and electroporation. After electroporation, the cells were aspirated from the electroporation cup and placed in 1 mL of 1640 medium (containing 10%FBS) . Transfer the cells to a 37℃, 5%CO₂ incubator, let stand for 30 minutes, then measure the cell density and adjust it to 4×10⁵ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) , transfer the cells to a 37℃, 5%CO₂ incubator for culture. Remarks: Each RNP was mixed with CD4+ and CD8+ T cells respectively before electroporation.

4. Culture of T cells after gene editing

4.1 On day 5, proliferation detection

Mix the β2M-KO or RFX5-GH-KO CD4+ or CD8+ T cells, sample 20 μL, use flow cytometry to detect the density of each cell, and supplement a certain volume of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to adjust its density to 4×10⁵ cells/mL.

4.2 On Day 8, proliferation detection

4.3 On Day 11, proliferation, knockout detection

Mix the β2M-KO or RFX5-GH-KO CD4+ or CD8+ T cells, sample 20 μL, use flow cytometry to detect the density and knockout efficiency of each cell, and supplement a certain volume of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to adjust the density to 4×10⁵ cells/mL.

4.4 On Day 14, proliferation detection and cryopreservation

Mix the β2M-KO or RFX5-GH-KO CD4+ or CD8+ T cells, sample 20 μL, and detect the density of each cell by flow cytometry. Transfer the cells of each group to a 50mL centrifuge tube, centrifuge at 300g for 5min, resuspend the cells in a freezing solution (90%FBS+10%DMSO) at a density of 4×10⁶/mL, and freeze in 1mL/tube. The cells were placed in a pre-freezing box, and the pre-freezing box was placed in a -80℃ refrigerator for 24 hours, and then transferred to a liquid nitrogen tank.

Purification, culture, and cryopreservation of NK cells

1. Day 0, Resuscitation of Cord Bloodor Peripheral Blood

Remove the cord blood or peripheral blood (allogenic or autologous) from the liquid nitrogen and shake it quickly in a 37℃ water bath until it completely melts. After the surface of the umbilical cord blood or peripheral blood bag was disinfected, it was transferred to a biological safety cabinet. The umbilical cord blood or peripheral blood was transferred from the blood bag to a 50mL centrifuge tube, and 1640 medium (10 times the volume of the cord blood or peripheral blood) was added. After mixing, centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 20mL of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to resuspend the cells, add T cell activation antibody, and transfer the cells to a 37℃, 5%CO² incubator Stand in the middle for 48h.

2. Day 2, Purification of NK Cells

After 48h, the cells in the culture flask were mixed and transferred to a 50mL centrifuge tube, centrifuged at 300g for 5min. After centrifugation, discard the supernatant, add 20mL MACS buffer to resuspend the cells, and centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 40 μL MACS CD56 immunomagnetic beads, mix well and incubate in the dark for 15 minutes, add 20 mL MACS buffer, centrifuge at 300 g for 5 minutes. Assemble the separation column of MACS and wipe the surface with alcohol to disinfect, and rinse the separation column with 3mL MACS buffer. After centrifugation, discard the supernatant, add 1mL MACS buffer to resuspend the cells, add the cells to the washed MACS separation column, and then rinse the separation column with MACS buffer twice, 3mL/time. After the MACS buffer has run out, get 10mL of cells in the lower layer, replace the centrifuge tube, remove the MACS sorting column, add 5mL of MACS buffer, use the booster to punch out the MACS buffer, and get 5mL of the upper layer of cells. Mix the upper and lower layers of cells separately, and sample 20 μL for flow cytometry staining and counting. The upper layer of cells is purified NK cells. Centrifuge the purified NK cells at 300g for 5min. After centrifugation, discard the supernatant and resuspend the NK cells with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) at a density of 5×10⁵ cells/mL, the cells were placed in a 37℃, 5%CO2 incubator for static culture.

3. Culture of NK cells

3.1 Day 5

Take the cells out of the incubator, carefully add 1 times the volume of the original medium 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) , and place the NK cells in the supplemented solution at 37℃, 5%CO² cultured in an incubator.

3.2 Day 8, proliferation detection

Mix NK cells, sample 20 μL, use flow cytometry to detect the density and purity of NK cells, and adjust the density to 5×10⁵/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) .

3.3 Day 11, Proliferation Detection

3.4 Day 14, proliferation detection and cryopreservation

Mix NK cells, take a sample of 20 μL, use flow cytometry to detect the density and purity of NK cells, transfer the cells to a 50mL centrifuge tube, centrifuge at 300g, 5min, and resuspend with freezing solution (90%FBS+10%DMSO) . The cell density was 6×10⁶ cells/mL, and 1mL/tube was frozen. The cells were placed in a pre-freezing box, and the pre-freezing box was placed in a -80℃ refrigerator for 24 hours, and then transferred to a liquid nitrogen tank.

Mixed culture of NK cells and immune tolerance T cells

The β2M and RFX5 genes of CD4+ and CD8+ T cells were knocked out by Crispr/Cas9 using β2M-sgRNA and RFX5-GH-sgRNA (as shown in Table 6) , respectively, and then amplified in vitro for 11 days, and the expression of HLA-ABC on the surface of T cells was detected by flow cytometry. In vitro, NK cells were mixed with β2M-KO or RFX5-KO CD4+ or CD8+ T cells at a ratio of 1: 1, and the HLA-ABC-negative and positive groups of T cells were detected on the 3rd and 6th days, respectively. The proportion and proliferation of cells.

1. NK Cell Density Adjustment

Mix the cultured NK cells, sample 20 μL, and detect the cell density by flow cytometry. Take out NK cells into a 50mL centrifuge tube, centrifuge at 300g for 5min, and adjust the cell density to 1×10⁶ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) .

2. Density adjustment of gene-edited T cells

Mix the gene-edited T cells separately, sample 20 μL, and detect the cell density by flow cytometry. Take out the T cells into a 50mL centrifuge tube, centrifuge at 300g for 5min, and adjust the cell density to 1×10⁶ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) .

3. Day 0, plating

The adjusted density of NK cells and T cells was mixed according to the ratio of 1: 1, cultured in 96U plate with a total volume of 200 μL, and the 96U plate was placed in a 37℃, 5%CO₂ incubator for static culture.

4. Day 3, Proliferation Assay

After the cells were mixed, 20 μL of the cell suspension was taken to detect the cell density and the negative and positive percentages of HLA-ABC by flow cytometry.

5. Day 6, Proliferation Assay

After mixing the cells, take 20 μL of the cell suspension and use a flow cytometer to detect the cell density and the negative and positive percentages of HLA-ABC

Experimental results

As shown in FIGs. 1A-1B, RFX5-KO umbilical cord blood CD4+ T cells exhibited increased survival and proliferation, as compared to corresponding β2M-KO umbilical cord blood CD4+ T cells, in the presence of autologous umbilical cord blood NK cells. The data demonstrates that RFX5-KO umbilical cord blood CD4+ T cells have better tolerance to autologous umbilical cord blood NK cells than the corresponding β2M-KO umbilical cord blood CD4+ T cells.

As shown in FIGs. 2A-2B, RFX5-KO umbilical cord blood CD8+ T cells exhibited increased survival and proliferation, as compared to corresponding β2M-KO umbilical cord blood CD8+ T cells, in the presence of autologous umbilical cord blood NK cells. The data demonstrates that RFX5-KO umbilical cord blood CD8+ T cells have better tolerance to autologous umbilical cord blood NK cells than the corresponding β2M-KO umbilical cord blood CD8+ T cells.

As shown in FIGs. 3A-3B, RFX5-KO umbilical cord blood CD4+ T cells exhibited increased survival and proliferation, as compared to corresponding β2M-KO umbilical cord blood CD4+ T cells, in the presence of allogeneic umbilical cord blood NK cells. The data demonstrates that RFX5-KO umbilical cord blood CD4+ T cells have better tolerance to allogeneic umbilical cord blood NK cells than the corresponding β2M-KO umbilical cord blood CD4+ T cells.

As shown in FIGs. 4A-4B, RFX5-KO peripheral blood CD4+ T cells exhibited increased survival and proliferation, as compared to corresponding β2M-KO peripheral blood CD4+ T cells, in the presence of autologous peripheral blood NK cells. The data demonstrates that RFX5-KO peripheral blood CD4+ T cells have better tolerance to autologous peripheral blood NK cells than the corresponding β2M-KO peripheral blood CD4+ T cells.

As shown in FIGs. 5A-5B, RFX5-KO peripheral blood CD8+ T cells exhibited increased survival and proliferation, as compared to corresponding β2M-KO peripheral blood CD8+ T cells, in the presence of autologous peripheral blood NK cells. The data demonstrates that RFX5-KO peripheral blood CD8+ T cells have better tolerance to autologous peripheral blood NK cells than the corresponding β2M-KO peripheral blood CD8+ T cells.

As shown in FIGs. 6A-6B, RFX5-KO umbilical cord blood CD8+ T cells exhibited increased survival and proliferation, as compared to corresponding β2M-KO umbilical cord blood CD8+ T cells, in the presence of allogeneic peripheral blood NK cells. The data demonstrates that RFX5-KO umbilical cord blood CD8+ T cells have better tolerance to allogeneic peripheral blood NK cells than the corresponding β2M-KO umbilical cord blood CD8+ T cells.

Example 3. Tolerance of RFX5-KO T cells to NK cells compared to RFXANK-KO, RFXAP-KO, CIITA-KO, and β2M-KO T cells

This example illustrates that RFX5-KO T cells have increased tolerance to NK cells, as compared to corresponding RFXANK-KO, RFXAP-KO, CIITA-KO, and β2M-KO T cells.

The β2M, RFX5, RFXANK, RFXAP, and CIITA genes of CD4+ and CD8+ T cells were knocked out by Crispr/Cas9, and the resulting cells were expanded in vitro for 11 days. The expression of HLA-ABC on the surface of the cells was detected by flow cytometry. In vitro, NK cells and RFXANK-KO, RFXAP-KO, CIITA-KO, or β2M-KO T cells were mixed in the ratio of 1: 1. Proportion and proliferation of HLA-ABC negative and positive T cells were detected on the 3rd day and the 6th day, respectively.

Preparation, culture, and cryopreservation of immune tolerance T cells are according to the protocol as described in Example 2, with crRNA or gRNA including those targeting β2M (β2M-sgRNA) , CIITA (CIITA-sgRNA) , RFX5 (RFX5-GH-sgRNA) , RFXANK (RFXANK-CD-sgRNA) , and RFXAP (RFXAP-CD-sgRNA) .

Purification, culture, and cryopreservation of autologous NK cells are according to the protocol as described in Example 2.

Mixed culture of NK cells and immune tolerance T cells is according to the protocol as described in Example 2.

Experimental Results

As shown in FIGs. 7A-7B, RFX5-GH-KO CD4+ T cells, RFXANK-CD-KO CD4+ T cells, and RFXAP-CD-KO CD4+ T cells exhibited the best, the second best, and the third best survival and proliferation, in the presence of NK cells. β2M-KO CD4+ T cells exhibited poor tolerance to NK cells. CIITA-AB-KO CD4+ T cells do not contain any HLA-ABC negative cells.

As shown in FIGs. 8A-8B, RFX5-GH-KO CD8+ T cells, RFXANK-CD-KO CD8+ T cells, and RFXAP-CD-KO CD8+ T cells exhibited the best, the second best, and the third best survival and proliferation, in the presence of NK cells. β2M-KO CD8+ T cells exhibited poor tolerance to NK cells. CIITA-AB-KO CD8+ T cells do not contain any HLA-ABC negative cells.

Example 4. Survival and proliferation of T cells prepared with different gRNA in the presence of NK cells

This example illustrates the effects of using different gRNAs on survival and proliferation of T cells in the presence of NK cells.

To knock out the RFX5 gene of CD4+ and CD8+ T cells, a total of 5 sgRNAs were synthesized, namely AB, CD, EF, GH, and IJ, as shown in Table 6. The T cells were gene-edited by Crispr/Cas9 technology using the sgRNAs, and then expanded in vitro for 11 days. The expression of HLA-ABC on the surface of T cells was detected by flow cytometry, and the sgRNA with the highest knockout efficiency was selected.

To knock out the RFXANK gene of CD4+ and CD8+ T cells, a total of 4 sgRNAs were synthesized, namely AB, CD, EF, and GH, as shown in Table 6. The T cells were gene-edited by Crispr/Cas9 technology using the sgRNAs, and then expanded in vitro for 11 days. The expression of HLA-ABC on the surface of T cells was detected by flow cytometry, and the sgRNA with the highest knockout efficiency was selected.

To knock out the RFXAP gene of CD4+ and CD8+ T cells, a total of 4 sgRNAs were synthesized, namely AB, CD, EF, and GH, as shown in Table 6. The T cells were gene-edited by Crispr/Cas9 technology using the sgRNAs, and then expanded in vitro for 11 days. The expression of HLA-ABC on the surface of T cells was detected by flow cytometry, and the sgRNA with the highest knockout efficiency was selected.

Preparation, culture, and cryopreservation of immune tolerance T cells are according to the protocol as described in Example 2.

Experimental results

As shown in FIGs. 9A-9B, CD4+ T cells with RFX5 gene edited by different sgRNAs exhibited different tolerance to NK cells: in the order from best to worst, RFX5-GH-KO, RFX5-CD-KO, RFX5-IJ-KO, RFX5-AB-KO, and RFX5-EF-KO. RFX5-EF-KO CD4+ T cells did not show any advantages over the corresponding β2M-KO CD4+ T cells.

As shown in FIGs. 10A-10B, CD8+ T cells with RFX5 gene edited by different sgRNAs exhibited different tolerance to NK cells: in the order from best to worst, RFX5-GH-KO, RFX5-CD-KO, RFX5-IJ-KO, RFX5-AB-KO, and RFX5-EF-KO. RFX5-EF-KO CD8+ T cells even did not show any advantageous over the corresponding β2M-KO CD8+ T cells.

As shown in FIGs. 11A-11B, CD4+ T cells with RFXANK gene edited by different sgRNAs exhibited different tolerance to NK cells-in the order from best to worst, RFXANK-CD-KO, RFXANK-AB-KO, RFXANK-EF-KO, and RFXANK-GH-KO.

As shown in FIGs. 12A-12B, CD8+ T cells with RFXANK gene edited by different sgRNAs exhibited different tolerance to NK cells-in the order from best to worst, RFXANK-CD-KO, RFXANK-AB-KO, RFXANK-EF-KO, and RFXANK-GH-KO.

As shown in FIGs. 13A-13B, CD4+ T cells with RFXAP gene edited by different sgRNAs exhibited different tolerance to NK cells-in the order from best to worst, RFXAP-CD-KO, RFXAP-EF-KO, RFXAP-GH-KO, and RFXAP-AB-KO.

As shown in FIGs. 14A-14B, CD8+ T cells with RFXAP gene edited by different sgRNAs exhibited different tolerance to NK cells-in the order from best to worst, RFXAP-CD-KO, RFXAP-EF-KO, RFXAP-GH-KO, and RFXAP-AB-KO.

As shown in FIG. 20A-20B, different gRNAs targeting RFX exhibited different efficiencies in knocking out HLA-ABC in CD4+ T cells and CD8+ T cells, respectively-in the order from the best to worst, GH, CD, IJ, AB, EF, and O.

As shown in FIG. 20C, different gRNAs targeting RFXANK exhibited different efficiencies in knocking out HLA-ABC in CD4+ T cells and CD8+ T cells, respectively-in the order from the best to worst, CD, AB, GH, EF, and O.

As shown in FIG. 20D, different gRNAs targeting RFXAP exhibited different efficiencies in knocking out HLA-ABC in CD4+ T cells and CD8+ T cells, respectively-in the order from the best to worst, CD, GH, EF, AB, and O.

As shown in FIG. 20E, knocking out CIITA does not knock out HLA-ABC in both CD4+ T cells and CD8+ T cells, respectively. Additionally, by comparing the best gRNA of each group in knocking out HLA-ABC, it was found that in CD4+ T cells, the gRNAs exhibits the following order in knockout efficiency (from best to worst) : RFX5-GH, RFXANK-CD, β2M, and RFXAP-CD; in CD8+T Cells, the gRNAs exhibits the following order in knockout efficiency (from best to worst) : RFX5-GH, RFXANK-CD, RFXAP-CD, and β2M.

Example 5. Tolerance of RFX5-KO CAR-T cells to allogenic T cells

Preparation, culture, and cryopreservation of universal CAR-T cells

1. Day 0, Resuscitation of Cord Blood

Remove the cord blood from the liquid nitrogen and shake it quickly in a 37℃ water bath until it completely melts. After the surface of the umbilical cord blood bag was disinfected, it was transferred to a biological safety cabinet. The umbilical cord blood was transferred from the blood bag to a 50mL centrifuge tube, and 1640 medium (10 times the volume of umbilical cord blood) was added. After mixing, centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 20mL of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to resuspend the cells, add T cell activation antibody, and transfer the cells to a 37℃, 5%CO₂ incubator to stand for 48h.

2. Day 2, Purification of T Cells

2.1 CD4+ T cells

After 48h, the cells in the culture flask were mixed and transferred to a 50mL centrifuge tube, centrifuged at 300g for 5min. After centrifugation, discard the supernatant, add 20mL MACS buffer to resuspend the cells, and centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 40 μL MACS CD4 immunomagnetic beads, mix well and incubate in the dark for 15 minutes, add 20 mL MACS buffer, centrifuge at 300 g for 5 minutes. Assemble the separation column of MACS and wipe the surface with alcohol to disinfect, and rinse the separation column with 3mL MACS buffer. After centrifugation, discard the supernatant, add 1mL MACS buffer to resuspend the cells, add the cells to the washed MACS separation column, and then rinse the separation column with MACS buffer twice, 3mL/time. After the MACS buffer has run out, get 10mL of lower layer cells, replace the centrifuge tube, remove the MACS sorting column, add 5mL of MACS buffer, use the booster to punch out the MACS buffer, and get 5mL of the upper layer cells. Mix the upper and lower layer cells separately, and sample 20 μL for flow cytometry staining and counting. The upper layer cells are purified CD4+ T cells.

2.2 CD8+ T cells

Centrifuge 10 mL of the CD4-sorted lower layer cells at 300 g for 5 min. After centrifugation, discard the supernatant, add 40 μL MACS CD8 immunomagnetic beads, mix well and incubate in the dark for 15 minutes, add 10 mL MACS buffer, centrifuge at 300 g for 5 minutes. Assemble the separation column of MACS and wipe the surface with alcohol to disinfect, and rinse the separation column with 3mL MACS buffer. After centrifugation, discard the supernatant, add 1mL MACS buffer to resuspend the cells, add the cells to the washed MACS separation column, and then rinse the separation column with MACS buffer twice, 3mL/time. After the MACS buffer has run out, get 10mL of lower layer cells, replace the centrifuge tube, remove the MACS sorting column, add 5mL of MACS buffer, use the booster to punch out the MACS buffer, and get 5mL of the upper layer cells. Mix the upper and lower layer cells separately, and sample 20 μL for flow cytometry staining and counting. The upper layer cells are purified CD8+ T cells.

3. On Day 2, Gene Editing of T Cells

3.1 Configure gRNA

Add 1 μL each of crRNA and tracrRNA to Nuclease-Free Duplex Buffer, with a total volume of 100 μL. See Table 5 for gRNA configuration and naming. Heat the gRNA at 95℃ for 5 minutes, take it out and let it stand at room temperature until its temperature returns to 20-25℃.

Table 5. gRNA Configuration and Grouping

3.2 Configure RNP

Add 1.5 μL of gRNA, 1.5 μL of Cas9, and 0.6 μL of Enhancer to 21.4 μL Opti-MEM, the total volume is 25 μL, and let the RNP stand at room temperature for 5 minutes.

3.3 Configuration of electroporation system and electroporation

Centrifuge the purified CD4+ and CD8+ T cells separately, 300g, 5min, resuspend in Opti-MEM, the resuspension density is 5～6×10⁶ cells/mL, take 25μL CD4+, CD8+T cell suspension and mix with 25μL RNP, transferred to the electroporation cup, set the electroporation parameters, voltage: 500V, pulse time: 20ms, and electroporation. After electroporation, the cells were aspirated from the electroporation cup and placed in 1 mL of 1640 medium (containing 10%FBS) . Transfer the cells to a 37℃, 5%CO₂ incubator, let stand for 30 minutes, then measure the cell density and adjust it to 4×10⁵ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) . Remarks: Each RNP was mixed with CD4+ and CD8+ T cells respectively before electroporation.

3.4 Virus transfection of T cells

The CAR19 lentivirus was added to the gene-edited T cells at 25 MOI to prepare universal CAR-T cells, and the cells were transferred to a 37℃, 5%CO₂ incubator for culture.

4. Universal CAR-T cell culture

4.1 Day 5, detection of proliferation, virus infection efficiency

Mix TCR-KO, β2M-KO, or RFX5-GH-KO CD4+ or CD8+ CAR-T cells. Take a sample of 20 μL, use a flow cytometer to detect the density of each cell and virus infection efficiency, and supplement a certain volume of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to adjust the density to 4×105 cells/mL.

4.2 Day 8, proliferation detection

Mix TCR-KO, β2M-KO, or RFX5-GH-KO CD4+ or CD8+ CAR-T cells, take a 20 μL sample, detect the density of each cell with a flow cytometer, and supplement a certain volume of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to adjust the density to 4×10⁵ cells/mL.

4.3 Day 11, Proliferation, Knockout Assay

Mix TCR-KO, β2M-KO, or RFX5-GH-KO CD4+ or CD8+ CAR-T cells, take a sample of 20 μL, use a flow cytometer to detect the density and knockout efficiency of each cell, and supplement a certain volume of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to adjust the density to 4×10⁵ cells/mL.

4.4 Day 14, proliferation, detection and cryopreservation

Mix TCR-KO, β2M-KO, or RFX5-GH-KO CD4+ or CD8+ CAR-T cells, sample 20 μL, and use a flow cytometer to detect the density of each cell. Transfer the cells of each group to a 50mL centrifuge tube, centrifuge at 300g for 5min, resuspend the cells in a freezing solution (90%FBS+10%DMSO) at a density of 4×10⁶/mL, and freeze in 1mL/tube. Place the cells in a pre-freezing box, put the pre-freezing box in a -80℃ refrigerator for 24 hours, and then transfer it to a liquid nitrogen tank

Culture and cryopreservation of allogeneic T cells

1. Day 0, Resuscitation of Cord Blood

Remove the cord blood from the liquid nitrogen and shake it quickly in a 37℃ water bath until it completely melts. After sterilizing the cord blood surface, transfer it to a biological safety cabinet, transfer the cord blood from the blood bag to a 50mL centrifuge tube, add 1640 medium (10 times the volume of the cord blood) , mix well and centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 20mL of 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to resuspend the cells, add T cell activation antibody, and transfer the cells to a 37℃, 5%CO₂ incubator Stand in the middle for 48h.

2. Day 2, Purification of T Cells

After 48h, the cells in the culture flask were mixed and transferred to a 50mL centrifuge tube, centrifuged at 300g for 5min. After centrifugation, discard the supernatant, add 20mL MACS buffer to resuspend the cells, and centrifuge at 300g for 5min. After centrifugation, discard the supernatant, add 30 μL MACS CD4 and CD8 immunomagnetic beads respectively, mix well and incubate in the dark for 15 minutes, add 20 mL MACS buffer, centrifuge at 300 g for 5 minutes. Assemble the separation column of MACS and wipe the surface with alcohol to disinfect, and rinse the separation column with 3mL MACS buffer. After centrifugation, discard the supernatant, add 1mL MACS buffer to resuspend the cells, add the cells to the washed MACS separation column, and then rinse the separation column with MACS buffer twice, 3mL/time. After the MACS buffer has run out, get 10mL of lower layer cells, replace the centrifuge tube, remove the MACS sorting column, add 5mL of MACS buffer, use the booster to punch out the MACS buffer, and get 5mL of the upper layer cells. Mix the upper and lower layer cells separately, and sample 20 μL for flow cytometry staining and counting. The upper layer cells are purified T cells. Centrifuge the upper layer cells at 300g for 5min. After centrifugation, discard the supernatant, adjust the density to 4×10⁵ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) , and transfer the cells to a 37℃, 5%CO₂ incubator cultured in a static medium.

3. Day 5, Proliferation Assay

Mix the cells, take a sample of 20 μL to detect the density of T cells by flow cytometry, adjust the density to 4×10⁵/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) , and transfer the cells to 37 ℃, 5%CO₂ incubator static culture.

4. Day 8, Proliferation Assay

5. Day 11, Proliferation Assay

6. Day 14, proliferation detection and cryopreservation

Mix the cells, take a 20 μL sample and measure the T cell density by flow cytometry. Transfer the cells to a 50mL centrifuge tube, centrifuge at 300g for 5min, resuspend the cells in a freezing solution (90%FBS+10%DMSO) at a density of 4×10⁶/mL, and freeze in 1mL/tube. The cells were placed in a pre-freezing box, and the pre-freezing box was placed in a -80℃ refrigerator for 24 hours, and then transferred to a liquid nitrogen tank.

Mixed culture of allogeneic T cells and universal CAR-T cells

1. Allogeneic T cell density adjustment

Mix the cultured T cells, sample 20 μL, and detect the cell density by flow cytometry. Take out the T cells into a 50mL centrifuge tube, centrifuge at 300g for 5min, and adjust the cell density to 4×10⁶ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) .

2. Density adjustment of universal CAR-T cells

The universal CAR-T cells cultured were mixed separately, 20 μL was sampled, and the cell density was detected by flow cytometry. Take out the universal CAR-T cells into a 50mL centrifuge tube, centrifuge at 300g for 5min, and adjust the cell density to 4×10⁶ cells/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) .

3. Day 0, plating

Mix allogeneic T cells with adjusted density and universal CAR-T cells at a ratio of 4: 1, 2: 1, and 1: 1, culture them in a 96U plate with a total volume of 200 μL, and place the 96U plate at 37℃ , cultured statically in a 5%CO2 incubator.

4. Day 3, Proliferation Assay

5. Day 6, Proliferation Assay

Experimental results

As shown in FIGs. 15A-16B, knocking out β2M or RFX5-GH can attenuate the immune rejection of CD4+ or CD8+ CAR-T cells by allogeneic T cells.

Example 6. Universal CAR-T cells kill tumor cells

1. CAR-T cell density adjustment

Mix and culture TCR or RFX5-GH knockout CD4+ or CD8+ CAR-T cells, or CD4+ or CD8+CAR-T cells without gene editing, respectively, and 20 μL was sampled, and the cell density was detected by flow cytometry. Take out the CAR-T cells into a 50mL centrifuge tube, centrifuge at 300g for 5min, and discard the supernatant. Use 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2) to adjust the cell density to 1×10⁶ cells/mL.

2. Tumor Cell Density Adjustment

Mix the Raji cells, sample 20 μL, and detect the cell density with a flow cytometer. Take out the Raji cells to a 50mL centrifuge tube, centrifuge at 300g for 5min, discard the supernatant. Adjust the cell density to 4×10⁶/mL with 1640 complete medium (containing 10%FBS and 1000IU/mL IL-2)

3. Day 0, plating

Mix the adjusted density of CAR-T cells and Raji cells according to the ratio of 1: 3, 1: 9, 1: 18, culture in a 48-well plate, the total volume is 500 μL, mix the cells, take 50 μL of cells to a 96U plate 5 μL of CD107a dye was added to each well, and the 48-well plate and 96U plate were placed in a 37℃, 5%CO² incubator for static culture.

4. CD107a detection

After 1 hour, the 96U plate was taken out, 7.5 μL monensin solution was added, and after mixing, the 96U plate was again placed in a 37℃, 5%CO₂ incubator for static culture. After 3 hours, the cells were aspirated from the 96U plate, and the expression of CD107a was detected by flow cytometry.

5. Day 3, kill assay

After the cells were mixed, 20 μL of the cell suspension was taken to measure the cell density with a flow cytometer to calculate the killing efficiency.

Experimental results

As shown in FIG. 17, knocking out RFX5-GH does not affect the virus transduction efficiency of CD4+ or CD8+ T cells.

As shown in FIG. 18, knocking out RFX5-GH did not affect the killing of Raji cells by CAR-T cells.

As shown in FIG. 19, knocking out RFX5-GH does not affect the expression of CD107a in CAR-T cells.

Numbered Embodiments

Embodiment 1. Apopulation of immune cells resulted from genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, wherein at least 70%of immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene.

Embodiment 2. The population of immune cells of embodiment 1, wherein the at least 70%of the immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene.

Embodiment 3. The population of immune cells of embodiment 1 or 2, wherein the genetic engineering does not knock out β2M gene, does not knock down β2M gene, or both.

Embodiment 4. The population of immune cells of any one of embodiments 1-3, wherein the population of immune cells does not have knockout of β2M gene.

Embodiment 5. The population of immune cells of any one of embodiments 1-4, wherein at least 70%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of the one or more of RFX5, RFXAP, or RFXANK protein.

Embodiment 6. The population of immune cells of any one of embodiments 1-5, wherein at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of one or more of RFX5, RFXAP, and RFXANK protein.

Embodiment 7. The population of immune cells of any one of embodiments 1-6, wherein at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene.

Embodiment 8. The population of immune cells of embodiment 7, wherein the one or more genomic alterations comprise deletion, insertion, substitution, or any combination thereof.

Embodiment 9. The population of immune cells of embodiment 7 or 8, wherein the one or more genomic alterations result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene.

Embodiment 10. The population of immune cells of any one of embodiments 1-9, wherein the population of immune cells comprises lymphocytes or myeloid cells.

Embodiment 11. The population of immune cells of embodiment 10, wherein the lymphocytes comprise T cells, B cells, tumor infiltrating lymphocytes, or natural killer cells.

Embodiment 12. The population of immune cells of embodiment 10 or 11, wherein the lymphocytes comprise CD8+ T cells or CD4+ T cells.

Embodiment 13. The population of immune cells of embodiment 11 or 12, wherein the T cells comprise a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .

Embodiment 14. The population of immune cells of any one of embodiments 10-13, wherein the lymphocytes comprise T cells, and the T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.

Embodiment 15. The population of immune cells of any one of embodiments 1-14, wherein the population of immune cells is derived from peripheral blood, bone marrow, placenta, or umbilical cord.

Embodiment 16. The population of immune cells of any one of embodiments 1-15, wherein the population of immune cells is derived from an autologous donor.

Embodiment 17. The population of immune cells of any one of embodiments 1-15, wherein the population of immune cells is derived from an allogenic donor.

Embodiment 18. The population of immune cells of any one of embodiments 1-17, wherein the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not knock out a NK activating receptor ligand gene in the immune cell.

Embodiment 19. The population of immune cells of any one of embodiments 1-18, wherein the population of immune cells does not have knockout of a NK activating receptor ligand gene, does not have knockdown of the NK activating receptor ligand gene, or both.

Embodiment 20. The population of immune cells of any one of embodiments 1-19, wherein the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cells.

Embodiment 21. The population of immune cells of any one of embodiments 1-20, wherein the population of immune cells does not have a heterologous nucleic acid encoding a NK inhibitory molecule.

Embodiment 22. The population of immune cells of any one of embodiments 1-21, wherein the percentage of the immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in the population is greater than the percentage of immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in a population of corresponding immune cells resulted from genetic engineering to knock out β2M gene.

Embodiment 23. The population of immune cells of embodiment 22, wherein the genetic engineering to knock out β2M gene does not knock out one or more of RFX5, RFXAP, or RFXANK gene.

Embodiment 24. The population of immune cells of embodiment 22 or 23, wherein the population of corresponding immune cells does not have knockout of one or more of RFX5, RFXAP, or RFXANK gene.

Embodiment 25. The population of immune cells of any one of embodiments 22-24, wherein the population of immune cells has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to the population of corresponding immune cells.

Embodiment 26. The population of immune cells of embodiment 25, wherein the NK cell-mediated cellular cytotoxicity is measured by an assay in which the population of immune cells is contacted with NK cells.

Embodiment 27. The population of immune cells of embodiment 26, wherein the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord.

Embodiment 28. The population of immune cells of any one of embodiments 22-27, wherein the population of immune cells has comparable tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.

Embodiment 29. The population of immune cell of any one of embodiments 22-28, wherein the population of immune cells has increased tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.

Embodiment 30. Apopulation of T cells comprising a CAR (CAR-T cells) , wherein:

(i) the CAR-T cells have one or more genomic alternations that reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein,

(ii) at least 70%of the CAR-T cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene,

(iii) the CAR-T cells do not have knockout of β2M gene, and

(iv) the CAR-T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.

Embodiment 31. The population of CAR-T cells of embodiment 30, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.

Embodiment 32. The population of CAR-T cells of embodiment 30, wherein the CAR-T cells have a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.

Embodiment 33. An immune cell that (i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein, and (ii) does not have knockout of β2M gene, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and/or HLA-C gene.

Embodiment 34. The immune cell of embodiment 33, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene.

Embodiment 35. The immune cell of embodiment 33 or 34, wherein the immune cell does not have knockdown of β2M gene.

Embodiment 36. The immune cell of embodiment 33 or 34, wherein the immune cell does not have knockout of β2M gene or knockdown of β2M gene.

Embodiment 37. The immune cell of any one of embodiments 33-36, wherein the one or more genomic alterations comprise one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene.

Embodiment 38. The immune cell of embodiment 37, wherein the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene comprise deletion, insertion, substitution, or any combination thereof.

Embodiment 39. The immune cell of embodiment 37 or 38, wherein the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene.

Embodiment 40. The immune cell of any one of embodiments 33-39, wherein the immune cell is a lymphocyte or a myeloid cell.

Embodiment 41. The immune cell of embodiment 40, wherein the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell.

Embodiment 42. The immune cell of embodiment 40 or 41, wherein the lymphocyte is a CD8+ T cell or a CD4+ T cell.

Embodiment 43. The immune cell of embodiment 41 or 42, wherein the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .

Embodiment 44. The immune cell of any one of embodiments 40-43, wherein the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.

Embodiment 45. The immune cell of embodiment 44, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.

Embodiment 46. The immune cell of embodiment 44, wherein the T cell has a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.

Embodiment 47. The immune cell of any one of embodiments 33-44, wherein the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord.

Embodiment 48. The immune cell of any one of embodiments 33-47, wherein the immune cell is derived from an autologous donor.

Embodiment 49. The immune cell of any one of embodiments 33-47, wherein the immune cell is derived from an allogenic donor.

Embodiment 50. The immune cell of any one of embodiments 33-49, wherein the immune cell does not have knockout of a NK activating receptor ligand gene, does not have knockdown of the NK activating receptor ligand gene, or both.

Embodiment 51. The immune cell of any one of embodiments 33-50, wherein the immune cell does not have a heterologous nucleic acid encoding a NK inhibitory molecule.

Embodiment 52. The immune cell of any one of embodiments 33-51, wherein the immune cell has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to a corresponding immune cell that has knockout of β2M gene.

Embodiment 53. The immune cell of embodiment 52, wherein the corresponding immune cell does not have one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein.

Embodiment 54. The immune cell of embodiment 52 or 53, wherein the NK cell-mediated cellular cytotoxicity is measured by an assay in which the immune cell is contacted with NK cells.

Embodiment 55. The immune cell of embodiment 54, wherein the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord.

Embodiment 56. The immune cell of any one of embodiments 52-55, wherein the immune cell has comparable tolerance to T cell-mediated cellular cytotoxicity, as compared to the corresponding immune cell.

Embodiment 57. AT cell comprising a CAR (CAR-T cell) , wherein the CAR-T cell

(i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein,

(ii) does not have knockout of β2M gene, and

(iii) has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.

Embodiment 58. The CAR-T cell of embodiment 57, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.

Embodiment 59. Amethod of reducing expression of HLA-A, HLA-B, or HLA-C gene while maintaining tolerance of an immune cell to host NK cells, comprising genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein in the immune cell.

Embodiment 60. The method of embodiment 59, wherein the method does not comprise knocking out β2M gene in the immune cell, does not comprise knocking down of the β2M gene in the immune cell, or both.

Embodiment 61. The method of embodiment 59 or 60, wherein the method does not comprise knocking out a NK activating receptor ligand gene in the immune cell, does not comprise knocking down the NK activating receptor ligand gene in the immune cell, or both.

Embodiment 62. The method of any one of embodiments 59-61, wherein the method does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cell.

Embodiment 63. The method of any one of embodiments 59-62, wherein the immune cell is a lymphocyte or a myeloid cell.

Embodiment 64. The method of embodiment 63, wherein the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell.

Embodiment 65. The method of embodiment 63 or 64, wherein the lymphocyte is a CD8+ T cell or a CD4+ T cell.

Embodiment 66. The method of embodiment 64 or 65, wherein the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .

Embodiment 67. The method of any one of embodiments 63-66, wherein the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.

Embodiment 68. The method of embodiment 67, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.

Embodiment 69. The method of any one of embodiments 59-68, wherein the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord.

Embodiment 70. The method of any one of embodiments 59-69, wherein the immune cells are derived from an autologous donor.

Embodiment 71. The method of any one of embodiments 59-69, wherein the immune cells are derived from an allogenic donor.

Embodiment 72. Amethod comprising administering the population of immune cells of any one of embodiments 1-29, the population of CAR-T cells of any one of embodiments 30-32, the immune cell of any one of embodiments 33-56, or the CAR-T cell of embodiment 57 or 58, to a subject in need thereof.

Embodiment 73. The method of embodiment 72, wherein the disease or condition is cancer, optionally acute lymphoblastic leukemia (ALL) .

Embodiment 74. Apharmaceutical composition comprising the population of immune cells of any one of embodiments 1-29, the population of CAR-T cells of any one of embodiments 30-32, the immune cell of any one of embodiments 33-56, or the CAR-T cell of embodiment 57 or 58, and a pharmaceutically acceptable excipient or carrier.

Embodiment 75. Use of the population of immune cells of any one of embodiments 1-29, the population of CAR-T cells of any one of embodiments 30-32, the immune cell of any one of embodiments 33-56, or the CAR-T cell of embodiment 57 or 58 in an adoptive cell therapy.

Embodiment 76. Aguide RNA (gRNA) or a polynucleotide encoding the guide RNA, the guide RNA comprising a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-16.

Embodiment 77. Aribonucleoprotein (RNP) complex comprising the gRNA of embodiment 76 and a Cas protein.

Embodiment 78. Acomposition comprising the gRNA of embodiment 76.

Embodiment 79. The composition of embodiment 78, wherein the composition comprises an immune cell that contains the gRNA.

Embodiment 80. The composition of embodiment 79, wherein the immune cell further comprises an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) .

Embodiment 81. Amethod of engineering a cell, comprising introducing the gRNA of embodiment 76 into the cell, or contacting the cell with the RNP complex of embodiment 77.

Embodiment 82. The method of embodiment 81, wherein the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-8; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-8, thereby resulting in genomic alteration of RFX5 gene in the cell.

Embodiment 83. The method of embodiment 81, wherein the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 9-12; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 9-12, thereby resulting in genomic alteration of RFXANK gene in the cell.

Embodiment 84. The method of embodiment 81, wherein the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 13-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 13-16, thereby resulting in genomic alteration of RFXAP gene in the cell.

Embodiment 85. The method of any one of embodiments 81-84, wherein the method comprises electroporating the cell with reagents comprising the gRNA or the RNP complex.

Embodiment 86. The method of any one of embodiments 81-85, wherein the method further comprises contacting the cell with a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) .

Embodiment 87. The method of embodiment 86, wherein the nucleic acid molecule is a vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

Embodiment 88. The method of any one of embodiments 81-87, wherein the cell is an immune cell, optionally a CD4+ T cell or a CD8+ T cell.

Embodiment 89. The method of any one of embodiments 81-88, wherein the cell is isolated from cord blood of a human or is a progeny of a cell isolated from cord blood of a human.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A population of immune cells resulted from genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein, wherein at least 70%of immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene.
The population of immune cells of claim 1, wherein the at least 70%of the immune cells in the population have reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene.
The population of immune cells of claim 1, wherein the genetic engineering does not knock out β2M gene, does not knock down β2M gene, or both.
The population of immune cells of claim 1, wherein the population of immune cells does not have knockout of β2M gene.
The population of immune cells of claim 1, wherein at least 70%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of the one or more of RFX5, RFXAP, or RFXANK protein.
The population of immune cells of any one of claims 1-5, wherein at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations that reduce or eliminate the expression or function of one or more of RFX5, RFXAP, and RFXANK protein.
The population of immune cells of any one of claims 1-5, wherein at least 80%, at least 90%, at least 95%, or about 100%of the immune cells in the population have one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene.
The population of immune cells of claim 7, wherein the one or more genomic alterations comprise deletion, insertion, substitution, or any combination thereof.
The population of immune cells of claim 7, wherein the one or more genomic alterations result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene.
The population of immune cells of any one of claims 1-5, wherein the population of immune cells comprises lymphocytes or myeloid cells.
The population of immune cells of claim 10, wherein the lymphocytes comprise T cells, B cells, tumor infiltrating lymphocytes, or natural killer cells.
The population of immune cells of claim 10, wherein the lymphocytes comprise CD8+ T cells or CD4+ T cells.
The population of immune cells of claim 11 or 12, wherein the T cells comprise a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .
The population of immune cells of claim 10, wherein lymphocytes comprise T cells, and the T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.
The population of immune cells of any one of claims 1-5, wherein the population of immune cells is derived from peripheral blood, bone marrow, placenta, or umbilical cord.
The population of immune cells of any one of claims 1-5, wherein the population of immune cells is derived from an autologous donor.
The population of immune cells of any one of claims 1-5, wherein the population of immune cells is derived from an allogenic donor.
The population of immune cells of any one of claims 1-5, wherein the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not knock out a NK activating receptor ligand gene in the immune cell.
The population of immune cells of any one of claims 1-5, wherein the population of immune cells does not have knockout of a NK activating receptor ligand gene, does not have knockdown of the NK activating receptor ligand gene, or both.
The population of immune cells of any one of claims 1-5, wherein the genetic engineering to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cells.
The population of immune cells of any one of claims 1-5, wherein the population of immune cells does not have a heterologous nucleic acid encoding a NK inhibitory molecule.
The population of immune cells of any one of claims 1-5, wherein the percentage of the immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in the population is greater than the percentage of immune cells that have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene in a population of corresponding immune cells resulted from genetic engineering to knock out β2M gene.
The population of immune cells of claim 22, wherein the genetic engineering to knock out β2M gene does not knock out one or more of RFX5, RFXAP, or RFXANK gene.
The population of immune cells of claim 22, wherein the population of corresponding immune cells does not have knockout of one or more of RFX5, RFXAP, or RFXANK gene.
The population of immune cells of claim 22, wherein the population of immune cells has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to the population of corresponding immune cells.
The population of immune cells of claim 25, wherein the NK cell-mediated cellular cytotoxicity is measured by an assay in which the population of immune cells is contacted with NK cells.
The population of immune cells of claim 26, wherein the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord.
The population of immune cells of claim 22, wherein the population of immune cells has comparable tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.
The population of immune cell of claim 22, wherein the population of immune cells has increased tolerance to T cell-mediated cellular cytotoxicity, as compared to the population of corresponding immune cells.
A population of T cells comprising a CAR (CAR-T cells) , wherein:

(i) the CAR-T cells have one or more genomic alternations that reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein,

(ii) at least 70%of the CAR-T cells in the population have reduced or eliminated expression of HLA-A, HLA-B, or HLA-C gene,

(iii) the CAR-T cells do not have knockout of β2M gene, and

(iv) the CAR-T cells have a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.
The population of CAR-T cells of claim 30, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.
The population of CAR-T cells of claim 30, wherein the CAR-T cells have a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.
An immune cell that (i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein, and (ii) does not have knockout of β2M gene, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and/or HLA-C gene.
The immune cell of claim 33, wherein the immune cell has reduced or eliminated expression of HLA-A, HLA-B, and HLA-C gene.
The immune cell of claim 33, wherein the immune cell does not have knockdown of β2M gene.
The immune cell of claim 33, wherein the immune cell does not have knockout of β2M gene or knockdown of β2M gene.
The immune cell of claim 33, wherein the one or more genomic alterations comprise one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene.
The immune cell of claim 37, wherein the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene comprise deletion, insertion, substitution, or any combination thereof.
The immune cell of claim 37, wherein the one or more genomic alterations in one or more of RFX5, RFXAP, and RFXANK gene result in a nonsense mutation of the one or more of RFX5, RFXAP, and RFXANK gene.
The immune cell of any one of claims 33-39, wherein the immune cell is a lymphocyte or a myeloid cell.
The immune cell of claim 40, wherein the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell.
The immune cell of claim 40, wherein the lymphocyte is a CD8+ T cell or a CD4+ T cell.
The immune cell of claim 41 or 42, wherein the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .
The immune cell of claim 40, wherein the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.
The immune cell of claim 44, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.
The immune cell of claim 44, wherein the T cell has a genomic alteration in a gene selected from the group consisting of: TRAC, TRBC, CD247, CD3G, CD3D, CD3E, and combinations thereof.
The immune cell of any one of claims 33-39, wherein the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord.
The immune cell of any one of claims 33-39, wherein the immune cell is derived from an autologous donor.
The immune cell of any one of claims 33-39, wherein the immune cell is derived from an allogenic donor.
The immune cell of any one of claims 33-39, wherein the immune cell does not have knockout of a NK activating receptor ligand gene, does not have knockdown of the NK activating receptor ligand gene, or both.
The immune cell of any one of claims 33-39, wherein the immune cell does not have a heterologous nucleic acid encoding a NK inhibitory molecule.
The immune cell of any one of claims 33-39, wherein the immune cell has increased tolerance to NK cell-mediated cellular cytotoxicity, compared to a corresponding immune cell that has knockout of β2M gene.
The immune cell of claim 52, wherein the corresponding immune cell does not have one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein.
The immune cell of claim 52, wherein the NK cell-mediated cellular cytotoxicity is measured by an assay in which the immune cell is contacted with NK cells.
The immune cell of claim 54, wherein the NK cells are in or derived from peripheral blood, bone marrow, placenta, or umbilical cord.
The immune cell of claim 52, wherein the immune cell has comparable tolerance to T cell-mediated cellular cytotoxicity, as compared to the corresponding immune cell.
A T cell comprising a CAR (CAR-T cell) , wherein the CAR-T cell

(i) has one or more genomic alterations that reduce or eliminate expression or function of one or more of RFX5, RFXAP, or RFXANK protein,

(ii) does not have knockout of β2M gene, and

(iii) has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.
The CAR-T cell of claim 57, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.
A method of reducing expression of HLA-A, HLA-B, or HLA-C gene while maintaining tolerance of an immune cell to host NK cells, comprising genetically modifying the immune cell to reduce or eliminate expression and/or function of one or more of RFX5, RFXAP, and RFXANK protein in the immune cell.
The method of claim 59, wherein the method does not comprise knocking out β2M gene in the immune cell, does not comprise knocking down of the β2M gene in the immune cell, or both.
The method of claim 59, wherein the method does not comprise knocking out a NK activating receptor ligand gene in the immune cell, does not comprise knocking down the NK activating receptor ligand gene in the immune cell, or both.
The method of claim 59, wherein the method does not comprise introducing a heterologous nucleic acid encoding a NK inhibitory molecule to the immune cell.
The method of any one of claims 59-62, wherein the immune cell is a lymphocyte or a myeloid cell.
The method of claim 63, wherein the lymphocyte is a T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer cell.
The method of claim 63, wherein the lymphocyte is a CD8+ T cell or a CD4+ T cell.
The method of claim 64 or 65, wherein the T cell comprises a native T cell receptor (TCR) , a non-native TCR, or a chimeric antigen receptor (CAR) .
The method of claim 63, wherein the lymphocyte is a T cell, and wherein the T cell has a genomic alteration that reduces or eliminates expression or function of a component of a TCR/CD3 complex.
The method of claim 67, wherein the component of a TCR/CD3 complex is one or more selected from TRAC, TRBC, CD3ζ, CD3γ, CD3δ, and CD3ε.
The method of any one of claims 59-62, wherein the immune cell is derived from peripheral blood, bone marrow, placenta, or umbilical cord.
The method of any one of claims 59-62, wherein the immune cells are derived from an autologous donor.
The method of any one of claims 59-62, wherein the immune cells are derived from an allogenic donor.
A method comprising administering the population of immune cells of any one of claims 1-5, the population of CAR-T cells of any one of claims 30-32, the immune cell of any one of claims 33-39, or the CAR-T cell of claim 57 or 58, to a subject in need thereof.
The method of claim 72, wherein the disease or condition is cancer, optionally acute lymphoblastic leukemia (ALL) .
A pharmaceutical composition comprising the population of immune cells of any one of claims 1-5, the population of CAR-T cells of any one of claims 30-32, the immune cell of any one of claims 33-39, or the CAR-T cell of claim 57 or 58, and a pharmaceutically acceptable excipient or carrier.
Use of the population of immune cells of any one of claims 1-5, the population of CAR-T cells of any one of claims 30-32, the immune cell of any one of claims 33-39, or the CAR-T cell of claim 57 or 58 in an adoptive cell therapy.
A guide RNA (gRNA) or a polynucleotide encoding the guide RNA, the guide RNA comprising a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-16.
A ribonucleoprotein (RNP) complex comprising the gRNA of claim 76 and a Cas protein.
A composition comprising the gRNA of claim 76.
The composition of claim 78, wherein the composition comprises an immune cell that contains the gRNA.
The composition of claim 79, wherein the immune cell further comprises an exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) .
A method of engineering a cell, comprising introducing the gRNA of claim 76 into the cell, or contacting the cell with the RNP complex of claim 77.
The method of claim 81, wherein the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 4-8; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 4-8, thereby resulting in genomic alteration of RFX5 gene in the cell.
The method of claim 81, wherein the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 9-12; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 9-12, thereby resulting in genomic alteration of RFXANK gene in the cell.
The method of claim 81, wherein the method comprises introducing into the cell the gRNA that comprises a sequence having (i) at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%sequence identity to any one of the sequences set forth in SEQ ID NOs: 13-16; or (ii) at most 4 nucleotide mutations relative to the sequence set forth in any one of SEQ ID NOs: 13-16, thereby resulting in genomic alteration of RFXAP gene in the cell.
The method of any one of claims 81-84, wherein the method comprises electroporating the cell with reagents comprising the gRNA or the RNP complex.
The method of any one of claims 81-84, wherein the method further comprises contacting the cell with a nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) .
The method of claim 86, wherein the nucleic acid molecule is a vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
The method of any one of claims 81-84, wherein the cell is an immune cell, optionally a CD4+ T cell or a CD8+ T cell.
The method of any one of claims 81-84, wherein the cell is isolated from cord blood of a human or is a progeny of a cell isolated from cord blood of a human.