Antibody resistance modified receptors to enhance cell-based therapies

Abstract

Methods of improving donor cell implantation in a subject thereof are provided. The methods may include providing a donor cell modified to express a first subtype of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), the first subtype being functionally indistinguishable, but immunological distinguishable, from a second subtype of the target protein, administering the donor cell to a subject, and administering the donor cell to the subject. The host cells in the subject are then selectively inhibited based on expression of the second subtype of the target protein, thereby improving implantation of the donor cells in the subject. Also provided are combinations for administration to a subject in need thereof, where the combination comprises (1) a population of cells modified to express a first subtype of a target protein (e.g., IL2RG), and (2) an antagonist that specifically binds to a second subtype of the target protein but does not specifically bind to the first subtype of the target protein (e.g., an anti-IL2RG antigen binding protein).

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Application No. 63 / 377,444, filed Sep. 28, 2022, and U.S. Application No. 63 / 578,729, filed Aug. 25, 2023, which are hereby incorporated by reference in their entireties for all purposes. Reference to Sequence Listing Submitted as an XML file via EFS WEB

[0002] The sequence listing written in the file 602716SEQLIST.xml is 177 kilobytes, created on Sep. 27, 2023, and is incorporated herein by reference. BACKGROUND OF THE INVENTION

[0003] Host conditioning is an important step prior to transplantation or adoptive cell therapy, in which an immunosuppressive agent is administered to the transplant recipient to support the engraftment of donor transplant cells. Conditioning therapy has several key objectives, including clearing the niche space for transplanted immune cells, suppressing allograft rejection, and depleting or reducing diseased cells (e.g., blood and solid tumors or defective immune cells). However, the standard-of-care agents for pre-transplant conditioning and post-transplant immunosuppression are genotoxic drugs, which cause damage to many tissues, leave patients highly immunosuppressed, and may induce secondary malignancies, thereby precluding many patients from receiving potentially curative transplant therapies.

[0004] Non-genotoxic lymphodepleting agents, such as monoclonal antibodies targeting lymphocytes, are an alternative conditioning therapy. However, one obstacle to using lymphodepleting agents as a conditioning therapy is the sensitivity of transplanted cells to their action in addition to the target host cells. SUMMARY OF THE INVENTION

[0005] Methods for improving engraftment of donor cells in a subject in need thereof are provided. Combinations or combination drugs for administration to a subject in need thereof are also provided. Isolated cells or cell populations modified to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG), which is different from a second isoform of IL2RG, are also provided. Methods for preparing the isolated cells or cell populations are also provided. Genetically engineered interleukin-2 receptor subunit gamma (IL2RG) proteins and nucleic acids encoding these proteins are also provided.

[0006] Methods are provided for improving engraftment of donor cells in a subject in need thereof. Some such methods include: (a) providing donor cells that have been modified to express a first subtype of a target protein, wherein the target protein is a protein expressed on the cell surface of hematopoietic cells, wherein the first subtype of the target protein is different from a second subtype of the target protein, and wherein the second subtype is expressed in host cells of the subject; (b) administering the donor cells to the subject; and (c) selectively inhibiting host cells in the subject based on their expression of the second subtype of the target protein, thereby improving engraftment of the donor cells in the subject.

[0007] In some such methods, the target protein is a receptor. In some such methods, the target protein is a cytokine receptor or a chemokine receptor, optionally wherein the target protein is a cytokine receptor. In some such methods, the target protein is a protein expressed on the cell surface of lymphocytes. In some such methods, the target protein is a subunit of a cytokine receptor interleukin-2 (IL-2) receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor, or IL-21 receptor. In some such methods, the target protein is interleukin-2 receptor subunit gamma (IL2RG).

[0008] In some such methods, the selective inhibition of host cells in step (c) does not include ablation of host cells or does not include ablation of host cells by an active killing mechanism. In some such methods, the selective inhibition in step (c) includes: (1) blocking growth of host cells to provide a competitive growth advantage to the donor cells; (2) blocking localization or trafficking of host cells to provide a competitive homing advantage to the donor cells; (3) blocking cell-cell interaction or adhesion of host cells to provide a competitive tissue infiltration advantage to the donor cells; or (4) blocking immune cell activation in host cells to provide a competitive advantage to the donor cells. In some such methods, the selective inhibition in step (c) includes blocking growth of host cells (i.e., blocking proliferation) and / or blocking immune cell activation in host cells to provide a competitive growth advantage to the donor cells.

[0009] In some such methods, the first and second subtypes are functionally indistinguishable but immunologically distinguishable. In some such methods, the donor cells express the first subtype of the target protein and the second subtype of the target protein. In some such methods, the donor cells express only the first subtype of the target protein.

[0010] In some such methods, a first subtype of a target protein is expressed from an expression vector in a donor cell. In some such methods, a genomic locus has been edited to express a first subtype of a target protein in a donor cell. In some such methods, the genomic locus is an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG and the genomic locus is the IL2RG genomic locus. In some such methods, the genomic locus is not an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG and the genomic locus is not the IL2RG genomic locus.

[0011] In some such methods, the first subtype of the target protein is a genetically engineered subtype of the target protein. In some such methods, the first subtype of the target protein is genetically engineered to contain a mutation, thereby providing an altered epitope, optionally, wherein the mutation is an artificial mutation. In some such methods, the altered epitope is a binding region of a target protein antagonist (such as an antigen-binding protein) such that the target protein antagonist exhibits a reduced or eliminated ability to bind and / or inhibit the first subtype of the target protein (e.g., compared to its ability to bind and / or inhibit the second subtype of the target protein). In some such methods, both the first subtype of the target protein and the second subtype of the target protein retain the ability to bind an endogenous ligand, optionally, wherein the target protein antagonist (such as an antigen-binding protein) blocks the binding of the endogenous ligand to the second subtype of the target protein but does not block the binding of the endogenous ligand to the first subtype of the target protein.

[0012] In some such methods, the target protein is IL2RG, the altered epitope is in the binding region of a target protein antagonist, the target protein antagonist is an antibody that comprises an immunoglobulin light chain or variable region thereof that comprises three light chain CDRs, and an immunoglobulin heavy chain or variable region thereof that comprises three heavy chain CDRs, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same. In some such methods, the target protein is IL2RG, and the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such methods, the target protein is IL2RG, and the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such methods, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147. In some such methods, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution. In some such methods, the target protein is IL2RG, and the mutation comprises an M145K substitution. In some such methods, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution, optionally wherein the mutation comprises a W90Q substitution. In some such methods, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0013] In some such methods, the selective inhibition in step (c) comprises administering a target protein antagonist to a subject, wherein the target protein antagonist specifically binds to a second subtype of the target protein but does not specifically bind to a first subtype of the target protein, optionally wherein step (c) comprises multiple administrations of the target protein antagonist. In some such methods, the target protein antagonist is an antigen-binding protein. In some such methods, the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

[0014] In some such methods, the target protein is IL2RG, and the antigen-binding protein comprises an immunoglobulin light chain or variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and the three heavy chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same. In some such methods, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same. In some such methods, the target protein is IL2RG, the antigen-binding protein comprises an immunoglobulin light chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consist essentially of or consist of the same, and the antigen-binding protein comprises an immunoglobulin heavy chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consist essentially of or consist of the same. In some such methods, the target protein is IL2RG, the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consist essentially of or consist of the same, and the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consist essentially of or consist of the same. In some such methods, the target protein is IL2RG, the antigen-binding protein comprises an immunoglobulin light chain, which comprises the sequence shown in SEQ ID NO: 20, consist essentially of or consist of the same, and the antigen-binding protein comprises an immunoglobulin heavy chain, which comprises the sequence shown in SEQ ID NO: 18, consist essentially of or consist of the same.

[0015] In some such methods, the donor cell and / or the host cell is a hematopoietic cell. In some such methods, the donor cell and / or the host cell is an immune cell. In some such methods, the donor cell and / or the host cell is a lymphocyte or lymphoid progenitor cell. In some such methods, the donor cell and / or the host cell is a T cell. In some such methods, the donor cell and / or the host cell is a tumor infiltrating lymphocyte (TIL). In some such methods, the donor cell and / or the host cell is a B cell. In some such methods, the donor cell and / or the host cell is a NK cell. In some such methods, the donor cell and / or the host cell is a hematopoietic stem and progenitor cell. In some such methods, the donor cell and / or the host cell is derived from a hematopoietic stem cell, or a hematopoietic stem and progenitor cell. In some such methods, the donor cell is derived from an induced pluripotent stem cell. In some such methods, the subject is a mammal or non-human mammal, and the donor cell is a mammalian cell or non-human mammalian cell. In some such methods, the subject is human, and the donor cell is a human cell.

[0016] In some such methods, the donor cell comprises or expresses a therapeutic molecule. In some such methods, the therapeutic molecule does not target a target protein. In some such methods, the donor cell comprises or expresses an immunoglobulin. In some such methods, the immunoglobulin does not target a target protein. In some such methods, the donor cell comprises a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR). In some such methods, the CAR or exogenous TCR does not target a target protein.

[0017] In some such methods, the donor cell is autologous. In some such methods, the donor cell is allogeneic or syngeneic.

[0018] In some such methods, the subject has a disease or disorder, and the method is used to treat the disease or disorder. In some such methods, the subject has cancer. In some such methods, the cancer is a solid tumor cancer. In some such methods, the cancer is a hematological cancer. In some such methods, the subject has a hematopoietic malignancy, and the method is used to treat the subject's hematopoietic malignancy. In some such methods, the subject has defective immune cells or a hematopoietic genetic defect. In some such methods, the hematopoietic genetic defect is sickle cell disease or severe combined immunodeficiency (SCID). In some such methods, the therapeutic molecule targets the diseased cells.

[0019] In some such methods, steps (b) and (c) occur simultaneously. In some such methods, step (b) occurs before step (c), optionally, wherein step (c) includes multiple administrations of the target protein antagonist after step (b). In some such methods, step (b) occurs after step (c), optionally, wherein step (c) includes multiple administrations of the target protein antagonist before step (b). In some such methods, step (c) occurs before and after step (b), optionally, wherein step (c) includes multiple administrations of the target protein antagonist before step (b) and / or multiple administrations of the target protein antagonist after step (b).

[0020] Some such methods further include generating donor cells by modifying a cell population to express a first subtype of the target protein prior to step (a). In some such methods, the cell population is a population of induced pluripotent stem cells, and the method further includes differentiating the induced pluripotent stem cells into the donor cells administered in step (a) prior to step (a), optionally, wherein the induced pluripotent stem cells differentiate into hematopoietic cells, lymphocytes or lymphoid progenitors, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem cells and progenitors. In some such methods, the cell population is a population of hematopoietic stem cells or a population of hematopoietic stem cells and progenitors, and the method further includes differentiating the hematopoietic stem cells or hematopoietic stem cells and progenitors into the donor cells administered in step (a) prior to step (a), optionally, wherein the hematopoietic stem cells or hematopoietic stem cells and progenitors differentiate into differentiated hematopoietic cells, lymphocytes or lymphoid progenitors, T cells, B cells or NK cells.

[0021] In some such methods, generating donor cells includes introducing an expression vector encoding a first subtype of the target protein to express the first subtype of the target protein prior to step (a). In some such methods, generating donor cells includes editing a genomic locus in the cell population to express a first subtype of the target protein prior to step (a). In some such methods, the genomic locus is an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is the IL2RG genomic locus. In some such methods, the genomic locus is not an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is not the IL2RG genomic locus.

[0022] In some such methods, editing includes introducing into a cell population: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in a genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the genomic locus and the exogenous donor nucleic acid inserts into or recombines with the genomic locus to generate a donor cell expressing a first subtype of a target protein. In some such methods, the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein; and (ii) a guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that is the nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the nuclease reagent comprises a Cas protein and a guide RNA, optionally, wherein the target protein is IL2RG, and the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 76-87, or the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 64-75, or optionally, wherein the target protein is IL2RG, and the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 136-153, or the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 118-135. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises homology arms. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN), optionally, wherein the target protein is IL2RG, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 88-117, or optionally, wherein the target protein is IL2RG, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 154-173.

[0023] In some such methods, the target protein is IL2RG, and (I) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:77, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:65, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:88-97; (II) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:83, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:71, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:98-107; (III) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:86, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:74, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:108-117; (IV) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:137, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:119, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:154-163; or (V) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:138, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:120, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:164-173.

[0024] Some such methods further comprise isolating a cell population from the subject or a different subject prior to modifying the cell population.

[0025] In another aspect, there is provided a combination or combination medicament for administration to a subject in need thereof. Some such combinations comprise: (a) a population of donor cells modified to express a first subtype of a target protein, wherein the target protein is a protein expressed on the cell surface of hematopoietic cells, wherein the first subtype of the target protein is different from the second subtype of the target protein; and (b) a target protein antagonist that specifically binds to the second subtype of the target protein but does not specifically bind to the first subtype of the target protein.

[0026] In some such combinations, the target protein is a receptor. In some such combinations, the target protein is a cytokine receptor or a chemokine receptor, optionally wherein the target protein is a cytokine receptor. In some such combinations, the target protein is a protein expressed on the cell surface of lymphocytes. In some such combinations, the target protein is a subunit of a cytokine receptor interleukin-2 (IL-2) receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor or IL-21 receptor. In some such combinations, the target protein is interleukin-2 receptor subunit gamma (IL2RG).

[0027] In some such combinations, the target protein antagonist selectively inhibits host cells in a subject based on the expression of a second subtype of its target protein. In some such combinations, the selective inhibition of host cells does not include ablating the host cells by an active killing mechanism. In some such combinations, the selective inhibition includes: (1) blocking the growth of the host cells to provide a competitive growth advantage for the donor cells; (2) blocking the localization or trafficking of the host cells to provide a competitive homing advantage for the donor cells; (3) blocking the cell-cell interactions or adhesion of the host cells to provide a competitive tissue infiltration advantage for the donor cells; or (4) blocking immune cell activation in the host cells to provide a competitive advantage for the donor cells. In some such combinations, the selective inhibition includes blocking the growth of the host cells (i.e., blocking proliferation) and / or blocking immune cell activation in the host cells to provide a competitive growth advantage for the donor cells.

[0028] In some such combinations, the first and second subtypes are functionally indistinguishable but immunologically distinguishable. In some such combinations, the donor cells express the first subtype of the target protein and the second subtype of the target protein. In some such combinations, the donor cells express only the first subtype of the target protein.

[0029] In some such combinations, the first subtype of the target protein is expressed from an expression vector in a population of donor cells. In some such combinations, the genomic locus is edited to express the first subtype of the target protein in a population of donor cells. In some such combinations, the genomic locus is an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG and the genomic locus is the IL2RG genomic locus. In some such combinations, the genomic locus is not an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG and the genomic locus is not the IL2RG genomic locus.

[0030] In some such combinations, the first subtype of the target protein is a genetically engineered subtype of the target protein. In some such combinations, the first subtype of the target protein is genetically engineered to contain a mutation that provides an altered epitope, optionally, wherein the mutation is an artificial mutation. In some such combinations, the altered epitope is the binding region of a target protein antagonist (e.g., an antigen-binding protein) such that the target protein antagonist exhibits reduced or eliminated ability to bind and / or inhibit the first subtype of the target protein (e.g., compared to its ability to bind and / or inhibit the second subtype of the target protein). In some such combinations, both the first subtype and the second subtype of the target protein retain the ability to bind an endogenous ligand, optionally, wherein the target protein antagonist (e.g., an antigen-binding protein) blocks the binding of the endogenous ligand to the second subtype of the target protein but does not block the binding of the endogenous ligand to the first subtype of the target protein.

[0031] In some such combinations, the target protein is IL2RG, the altered epitope is in the binding region of a target protein antagonist, the target protein antagonist is an antibody that comprises an immunoglobulin light chain or a variable region thereof that comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof that comprises three heavy chain CDRs, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same.

[0032] In some such combinations, the target protein is IL2RG, and the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such combinations, the target protein is IL2RG, and the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such combinations, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147. In some such combinations, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution. In some such combinations, the target protein is IL2RG, and the mutation comprises an M145K substitution. In some such combinations, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution, optionally wherein the mutation comprises a W90Q substitution. In some such combinations, the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0033] In some such combinations, the target protein antagonist is an antigen-binding protein. In some such combinations, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such combinations, the target protein is IL2RG, and the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences shown in SEQ ID NOs: 12, 14, and 16, and the three heavy chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences shown in SEQ ID NOs: 4, 6, and 8. In some such combinations, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences shown in SEQ ID NOs: 12, 14, and 16, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences shown in SEQ ID NOs: 4, 6, and 8. In some such combinations, the target protein is IL2RG, and the antigen-binding protein comprises an immunoglobulin light chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence shown in SEQ ID NO: 10, and the antigen-binding protein comprises an immunoglobulin heavy chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence shown in SEQ ID NO: 2. In some such combinations, the target protein is IL2RG, the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence shown in SEQ ID NO: 10, and the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence shown in SEQ ID NO: 2. In some such combinations, the target protein is IL2RG, and the antigen-binding protein comprises an immunoglobulin light chain, which comprises the sequence shown in SEQ ID NO: 20, consists essentially of or consists of the sequence shown in SEQ ID NO: 20, and the antigen-binding protein comprises an immunoglobulin heavy chain, which comprises the sequence shown in SEQ ID NO: 18, consists essentially of or consists of the sequence shown in SEQ ID NO: 18.

[0034] In some such combinations, the donor cell is a hematopoietic cell. In some such combinations, the donor cell is an immune cell. In some such combinations, the donor cell is a lymphocyte or a lymphoid progenitor cell. In some such combinations, the donor cell is a T cell. In some such combinations, the donor cell is a tumor-infiltrating lymphocyte (TIL). In some such combinations, the donor cell is a B cell. In some such combinations, the donor cell is an NK cell. In some such combinations, the donor cell is a hematopoietic stem cell, or a hematopoietic stem cell and progenitor cells. In some such combinations, the donor cell is derived from induced pluripotent stem cells, or from hematopoietic stem cells, or from hematopoietic stem cells and progenitor cells.

[0035] In some such combinations, the subject is a mammal or a non - human mammal, and the donor cell is a mammalian cell or a non - human mammalian cell. In some such combinations, the subject is human, and the donor cell is a human cell.

[0036] In some such combinations, the donor cell contains or expresses a therapeutic molecule. In some such combinations, the therapeutic molecule does not target a target protein. In some such combinations, the donor cell contains or expresses an immunoglobulin. In some such combinations, the immunoglobulin does not target a target protein. In some such combinations, the donor cell contains a chimeric antigen receptor (CAR) or an exogenous T - cell receptor (TCR). In some such combinations, the CAR or the exogenous TCR does not target a target protein.

[0037] In some such combinations, the donor cell is autologous. In some such combinations, the donor cell is allogeneic or syngeneic.

[0038] In some such combinations, the subject has a disease or disorder, and the combination is used to treat the disease or disorder. In some such combinations, the subject has cancer. In some such combinations, the cancer is a solid - tumor cancer. In some such combinations, the cancer is a hematological cancer. In some such combinations, the subject has a hematopoietic malignancy, and the combination therapy is used to treat the subject's hematopoietic malignancy. In some such combinations, the subject has defective immune cells or a hematopoietic genetic defect. In some such combinations, the hematopoietic genetic defect is sickle - cell disease or severe combined immunodeficiency (SCID). In some such combinations, the therapeutic molecule targets diseased cells.

[0039] On the other hand, there are provided isolated cells or cell populations that have been modified to express a first subtype of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second subtype of IL2RG. In some such cells or populations, the first subtype of IL2RG has been genetically engineered to contain a mutation, thereby providing an altered epitope that is the binding region for an IL2RG antagonist (e.g., an antigen-binding protein), such that the IL2RG antagonist exhibits a reduced or eliminated ability to bind to and / or inhibit the first subtype of IL2RG (e.g., compared to its ability to bind to and / or inhibit the second subtype of IL2RG), and the first subtype of IL2RG retains the ability to bind to its endogenous ligand. In some such cells or populations, the mutation is an artificial mutation. In some such cells or populations, both the first subtype of IL2RG and the second subtype of IL2RG retain the ability to bind to the endogenous ligand, optionally, wherein the IL2RG antagonist (e.g., an antigen-binding protein) blocks the binding of the endogenous ligand to the second subtype of IL2RG but does not block the binding of the endogenous ligand to the first subtype of IL2RG.

[0040] In some such cells or populations, the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable. In some such cells or populations, one or more cells express both the first subtype of IL2RG and the second subtype of IL2RG. In some such cells or populations, one or more cells express only the first subtype of IL2RG.

[0041] In some such cells or populations, the first subtype of IL2RG is expressed in one or more cells from an expression vector. In some such cells or populations, the genomic locus has been edited to express the first subtype of IL2RG in one or more cells. In some such cells or populations, the genomic locus is the endogenous IL2RG genomic locus. In some such cells or populations, the genomic locus is not the endogenous IL2RG genomic locus.

[0042] In some such cells or populations, the altered epitope is in the binding region of an IL2RG antagonist that is an antibody comprising an immunoglobulin light chain or its variable region that comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region that comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO:12, 14, and 16, consist essentially of or consist of the same, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO:4, 6, and 8, consist essentially of or consist of the same.

[0043] In some such cells or populations, the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such cells or populations, the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such cells or populations, the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147. In some such cells or populations, the mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution. In some such cells or populations, the mutation comprises an M145K substitution. In some such cells or populations, the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution, optionally wherein the mutation comprises a W90Q substitution. In some such cells or populations, the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0044] In some such cells or populations, the first and second subtypes can be immunologically distinguished by an IL2RG antagonist that specifically binds to the second subtype of IL2RG but not to the first subtype of IL2RG. In some such cells or populations, the IL2RG antagonist is an antigen-binding protein. In some such cells or populations, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such cells or populations, the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof that comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof that comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences, and the three heavy chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences. In some such cells or populations, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences. In some such cells or populations, the antigen-binding protein comprises an immunoglobulin light chain variable region that comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence, and the antigen-binding protein comprises an immunoglobulin heavy chain variable region that comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence. In some such cells or populations, the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence, and the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence. In some such cells or populations, the antigen-binding protein comprises an immunoglobulin light chain that comprises the sequence shown in SEQ ID NO: 20, consists essentially of or consists of the sequence, and the antigen-binding protein comprises an immunoglobulin heavy chain that comprises the sequence shown in SEQ ID NO: 18, consists essentially of or consists of the sequence.

[0045] In some such cells or populations, one or more cells are hematopoietic cells. In some such cells or populations, one or more cells are immune cells. In some such cells or populations, one or more cells are lymphocytes or lymphoid progenitor cells. In some such cells or populations, one or more cells are T cells. In some such cells or populations, one or more cells are tumor-infiltrating lymphocytes (TIL). In some such cells or populations, one or more cells are B cells. In some such cells or populations, one or more cells are NK cells. In some such cells or populations, one or more cells are hematopoietic stem cells, or hematopoietic stem cells and progenitor cells. In some such cells or populations, one or more cells are induced pluripotent stem cells. In some such cells or populations, one or more cells are mammalian cells or non-human mammalian cells. In some such cells or populations, one or more cells are human cells.

[0046] In some such cells or populations, one or more cells contain or express a therapeutic molecule. In some such cells or populations, the therapeutic molecule does not target IL2RG. In some such cells or populations, one or more cells contain or express an immunoglobulin. In some such cells or populations, the immunoglobulin does not target IL2RG. In some such cells or populations, one or more cells contain a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR). In some such cells or populations, the CAR or exogenous TCR does not target IL2RG.

[0047] In some such cells or populations, one or more cells are isolated from a subject. In some such cells or populations, one or more cells are used to treat a subject having cells expressing a second subtype of IL2RG.

[0048] On the other hand, methods for preparing any of the above-separated cells or cell populations are provided. Some such methods include modifying the cell or cell population to express a first isoform of IL2RG. In some such methods, the modification includes introducing an expression vector encoding the first isoform of IL2RG. In some such methods, the modification includes editing a genomic locus to express the first isoform of IL2RG. In some such methods, the genomic locus is the endogenous IL2RG genomic locus. In some such methods, the genomic locus is not the endogenous IL2RG genomic locus. In some such methods, the editing includes introducing into the cell: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in the genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the genomic locus, and the exogenous donor nucleic acid inserts into or recombines with the genomic locus to produce a donor cell expressing the first isoform of IL2RG. In some such methods, the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein; and (ii) a guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that serves as a nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the nuclease reagent comprises a Cas protein and a guide RNA, optionally, wherein the target protein is IL2RG, and the DNA targeting fragment comprises a sequence shown in any of SEQ ID NOs: 76-87, or the guide RNA target sequence comprises a sequence shown in any of SEQ ID NOs: 64-75, or optionally, wherein the target protein is IL2RG, and the DNA targeting fragment comprises a sequence shown in any of SEQ ID NOs: 136-153, or the guide RNA target sequence comprises a sequence shown in any of SEQ ID NOs: 118-135. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises homology arms. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN), optionally, wherein the target protein is IL2RG, and the ssODN comprises a nucleic acid sequence shown in any of SEQ ID NOs: 88-117, or optionally, wherein the target protein is IL2RG, and the ssODN comprises a nucleic acid sequence shown in any of SEQ ID NOs: 154-173.

[0049] In some such methods, the target protein is IL2RG, and (I) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:77, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:65, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:88-97; (II) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:83, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:71, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:98-107; (III) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:86, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:74, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:108-117; (IV) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:137, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:119, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:154-163; or (V) the DNA targeting fragment comprises the sequence shown in SEQ ID NO:138, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:120, and the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs:164-173.

[0050] In another aspect, a genetically engineered human interleukin-2 receptor subunit gamma (IL2RG) protein is provided. In some such genetically engineered IL2RG proteins, the protein comprises an artificial mutation to provide an altered epitope, the altered epitope being the binding region of an IL2RG antagonist (such as an antigen-binding protein), such that the IL2RG antagonist exhibits reduced or eliminated ability to bind and / or inhibit the genetically engineered IL2RG protein (e.g., compared to its ability to bind and / or inhibit the wild-type human IL2RG protein), and the genetically engineered IL2RG protein retains its binding to its endogenous ligand. In some such genetically engineered IL2RG proteins, the genetically engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable but immunologically distinguishable.

[0051] In some such genetically engineered IL2RG proteins, the altered epitope is in the binding region of an IL2RG antagonist, which is an antibody that comprises an immunoglobulin light chain or its variable region, which comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy chain CDRs, the three light chain CDRs respectively comprising the sequences shown in SEQ ID NO: 12, 14, and 16, consisting essentially of or consisting of the same, and the three heavy chain CDRs respectively comprising the sequences shown in SEQ ID NO: 4, 6, and 8, consisting essentially of or consisting of the same.

[0052] In some such genetically engineered IL2RG proteins, the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such genetically engineered IL2RG proteins, the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such genetically engineered IL2RG proteins, the engineered mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147. In some such genetically engineered IL2RG proteins, the engineered mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution. In some such genetically engineered IL2RG proteins, the engineered mutation comprises an M145K substitution. In some such genetically engineered IL2RG proteins, the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution, optionally wherein the mutation comprises a W90Q substitution. In some such genetically engineered IL2RG proteins, the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0053] In some such genetically engineered IL2RG proteins, the genetically engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable, but are immunologically distinguishable by an IL2RG antagonist. In some such genetically engineered IL2RG proteins, the IL2RG antagonist is an antigen-binding protein. In some such genetically engineered IL2RG proteins, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such genetically engineered IL2RG proteins, the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences, and the three heavy chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences. In some such genetically engineered IL2RG proteins, the three light chain CDRs each comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences, and the three heavy chain CDRs each comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences. In some such genetically engineered IL2RG proteins, the antigen-binding protein comprises an immunoglobulin light chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence, and the antigen-binding protein comprises an immunoglobulin heavy chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence. In some such genetically engineered IL2RG proteins, the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence, and the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence. In some such genetically engineered IL2RG proteins, the antigen-binding protein comprises an immunoglobulin light chain, which comprises the sequence shown in SEQ ID NO: 20, consists essentially of or consists of the sequence, and the antigen-binding protein comprises an immunoglobulin heavy chain, which comprises the sequence shown in SEQ ID NO: 18, consists essentially of or consists of the sequence.

[0054] In another aspect, provided are nucleic acids encoding any of the above-described genetically engineered IL2RG proteins. Some such nucleic acids are expression vectors encoding a genetically engineered human IL2RG protein.

[0055] Methods are provided for in vivo selectively depleting unedited cells in a subject. Such methods can include providing cells edited to express a first isoform of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), administering the edited cells to a subject, and then selectively depleting unedited cells in the subject based on the expression of a second isoform of the target protein. Such methods can include providing cells edited to express a first isoform of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), the first isoform being functionally indistinguishable but immunologically distinguishable from a second isoform of the target protein, administering the edited cells to a subject, and then selectively depleting unedited cells in the subject based on the expression of a second isoform of the target protein. Combinations for administration to a subject in need are also provided, wherein the combination comprises (1) a population of cells edited to express a first isoform of a target protein (e.g., IL2RG), and (2) an antagonist (e.g., an anti-IL2RG antigen-binding protein) that specifically binds the second isoform of the target protein but does not specifically bind the first isoform of the target protein. Isolated cells or cell populations modified to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second isoform of IL2RG are also provided. Isolated cells or cell populations are also provided, wherein the genomic locus has been edited to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second isoform of IL2RG. Isolated cells or cell populations are also provided, wherein the IL2RG genomic locus has been edited to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second isoform of IL2RG, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable. Methods for preparing isolated cells or cell populations are also provided. Genetically engineered interleukin-2 receptor subunit gamma (IL2RG) proteins and nucleic acids encoding such proteins are also provided.

[0056] In one aspect, methods are provided for in vivo selectively depleting unedited cells in a subject in need thereof. Some such methods include: (a) providing edited cells in which the IL2RG genomic locus has been edited to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second isoform of IL2RG, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable, and wherein the second isoform is expressed in the unedited cells of the subject; (b) administering the edited cells to the subject; and (c) selectively depleting the unedited cells in the subject based on the expression of the second isoform of IL2RG. In one aspect, methods are provided for in vivo selectively depleting unedited cells in a subject in need thereof and repopulating with edited cells. Some such methods include: (a) providing edited cells that have been modified to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second isoform of IL2RG, and wherein the second isoform is expressed in the unedited cells of the subject; (b) administering the edited cells to the subject; and (c) selectively depleting the unedited cells in the subject based on the expression of the second isoform of IL2RG. In some such methods, the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable. In some such methods, the edited cells express the first isoform of IL2RG and the second isoform of IL2RG. In some such methods, the edited cells express only the first isoform of IL2RG.

[0057] In some such methods, the first isoform of IL2RG is a genetically engineered isoform of IL2RG. In some such methods, the first isoform of IL2RG is genetically engineered to contain a mutation, thereby providing an altered epitope, optionally, wherein the mutation is an artificial mutation.

[0058] In some such methods, the altered epitope is in the binding region of an antibody that comprises an immunoglobulin light chain or a variable region thereof that comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof that comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same.

[0059] In some such methods, the mutation comprises a mutation in the nucleotide encoding within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such methods, the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such methods, the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143 or position K147. In some such methods, the mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution or an M145Y substitution. In some such methods, the mutation comprises an M145K substitution. In some such methods, the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution or a W90D substitution. In some such methods, the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0060] In some such methods, the selective depletion in step (c) comprises administering an IL2RG antagonist to a subject, wherein the IL2RG antagonist specifically binds to a second isoform of IL2RG but does not specifically bind to a first isoform of IL2RG. In some such methods, the IL2RG antagonist is an antigen-binding protein. In some such methods, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such methods, the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof that comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof that comprises three heavy chain CDRs, wherein the three light chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences shown in SEQ ID NOs: 12, 14, and 16, and wherein the three heavy chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences shown in SEQ ID NOs: 4, 6, and 8. In some such methods, the three light chain CDRs each comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences shown in SEQ ID NOs: 12, 14, and 16, and the three heavy chain CDRs each comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences shown in SEQ ID NOs: 4, 6, and 8. In some such methods, the immunoglobulin light chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence shown in SEQ ID NO: 10, and the immunoglobulin heavy chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence shown in SEQ ID NO: 2. In some such methods, the immunoglobulin light chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence shown in SEQ ID NO: 10, and the immunoglobulin heavy chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence shown in SEQ ID NO: 2.

[0061] In some such methods, the edited cells are hematopoietic cells. In some such methods, the edited cells are lymphocytes or lymphoid progenitor cells. In some such methods, the edited cells are T cells. In some such methods, the edited cells are tumor-infiltrating lymphocytes (TILs). In some such methods, the edited cells are B cells. In some such methods, the edited cells are NK cells. In some such methods, the edited cells are hematopoietic stem and progenitor cells. In some such methods, the edited cells are derived from induced pluripotent stem cells. In some such methods, the edited cells are derived from hematopoietic stem cells, or hematopoietic stem and progenitor cells. In some such methods, the subject is a mammal or non-human mammal, and the edited cells are mammalian cells or non-human mammalian cells. In some such methods, the subject is human, and the edited cells are human cells. In some such methods, the edited cells contain or express a therapeutic molecule. In some such methods, the edited cells contain or express an immunoglobulin. In some such methods, the edited cells contain a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR). In some such methods, the edited cells are autologous. In some such methods, the edited cells are allogeneic or syngeneic.

[0062] In some such methods, the subject has a hematopoietic malignancy, and the method is used to treat the subject's hematopoietic malignancy. In some such methods, the subject has cancer. In some such methods, the cancer is a hematological cancer. In some such methods, the subject has defective immune cells or a hematopoietic genetic defect. In some such methods, the hematopoietic genetic defect is sickle cell disease or severe combined immunodeficiency (SCID).

[0063] In some such methods, steps (b) and (c) occur simultaneously. In some such methods, step (b) occurs before step (c). In some such methods, step (b) occurs after step (c).

[0064] Some such methods also include generating edited cells by modifying a cell population to express a first isoform of IL2RG prior to step (a). In some such methods, the cell population is a population of induced pluripotent stem cells, and the method further includes differentiating the edited induced pluripotent stem cells into the edited cells administered in step (a) prior to step (a), optionally, wherein the induced pluripotent stem cells differentiate into hematopoietic cells, lymphocytes or lymphoid progenitors, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem cells and progenitors. In some such methods, the cell population is a population of hematopoietic stem cells, or a population of hematopoietic stem cells and progenitors, and the method further includes differentiating the edited hematopoietic stem cells or hematopoietic stem cells and progenitors into the edited cells administered in step (a) prior to step (a), optionally, wherein the hematopoietic stem cells or hematopoietic stem cells and progenitors differentiate into differentiated hematopoietic cells, lymphocytes or lymphoid progenitors, T cells, B cells or NK cells.

[0065] In some such methods, the genomic locus has been edited to express a first isoform of IL2RG in the edited cells. In some such methods, the genomic locus is the IL2RG genomic locus. In some such methods, the genomic locus is not the IL2RG genomic locus. In some such methods, the method further includes generating edited cells by editing the genomic locus in a cell population to express a first isoform of IL2RG prior to step (a). In some such methods, the method further includes generating edited cells by editing the IL2RG genomic locus in a cell population to express a first isoform of IL2RG prior to step (a). In some such methods, the cell population is a population of induced pluripotent stem cells, the IL2RG locus is edited in the induced pluripotent stem cells to produce edited induced pluripotent stem cells that express a first isoform of IL2RG, and the method further includes differentiating the edited induced pluripotent stem cells into the edited cells administered in step (a) prior to step (a), optionally, wherein the induced pluripotent stem cells differentiate into hematopoietic cells, lymphocytes or lymphoid progenitors, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem cells and progenitors. In some such methods, the cell population is a population of hematopoietic stem cells, or a population of hematopoietic stem cells and progenitors, the IL2RG locus is edited in the hematopoietic stem cells or hematopoietic stem cells and progenitors to produce edited hematopoietic stem cells or hematopoietic stem cells and progenitors that express a first isoform of IL2RG, and the method further includes differentiating the edited hematopoietic stem cells or hematopoietic stem cells and progenitors into the edited cells administered in step (a) prior to step (a), optionally, wherein the hematopoietic stem cells or hematopoietic stem cells and progenitors differentiate into differentiated hematopoietic cells, lymphocytes or lymphoid progenitors, T cells, B cells or NK cells.

[0066] In some such methods, the editing comprises introducing into a cell population: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in a genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the genomic locus and the exogenous donor nucleic acid inserts into or recombines with the genomic locus to produce an edited cell that expresses a first isoform of IL2RG. In some such methods, the editing comprises introducing into a cell population: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in the IL2RG genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the IL2RG genomic locus and the exogenous donor nucleic acid inserts into or recombines with the IL2RG genomic locus to produce an edited cell that expresses a first isoform of IL2RG. In some such methods, the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein; and (ii) a guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that serves as the nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the nuclease reagent comprises a Cas protein and a guide RNA, optionally wherein the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 76-87, or optionally wherein the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 64-75. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises homology arms. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN), optionally wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 88-117.

[0067] In some such methods, the method further comprises isolating the cell population from the subject or a different subject prior to modifying the cell population. In some such methods, the method further comprises isolating the cell population from the subject or a different subject prior to editing the IL2RG genomic locus.

[0068] In another aspect, there is provided a combination or combination medicament for administration to a subject in need. In some such combinations, the combination comprises: (a) a cell population modified to express a first subtype of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second subtype of IL2RG; and (b) an IL2RG antagonist that specifically binds to the second subtype of IL2RG but does not specifically bind to the first subtype of IL2RG. In some such combinations, the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable. In some such combinations, the genomic locus has been edited to express the first subtype of IL2RG in the cell population. In some such combinations, the genomic locus is the IL2RG genomic locus. In some such combinations, the genomic locus is not the IL2RG genomic locus. In some such combinations, the combination comprises: (a) a cell population in which the IL2RG genomic locus has been edited to express a first subtype of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second subtype of IL2RG, wherein the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable; and (b) an IL2RG antagonist that specifically binds to the second subtype of IL2RG but does not specifically bind to the first subtype of IL2RG. In some such combinations, the cells express the first subtype of IL2RG and the second subtype of IL2RG. In some such combinations, the cells express only the first subtype of IL2RG.

[0069] In some such combinations, the first subtype of IL2RG is a genetically engineered subtype of IL2RG. In some such combinations, the first subtype of IL2RG is genetically engineered to contain a mutation, thereby providing an altered epitope, optionally, wherein the mutation is an artificial mutation. In some such combinations, the altered epitope is in the binding region of an antibody that comprises an immunoglobulin light chain or its variable region, which comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same.

[0070] In some such combinations, the mutation comprises a mutation in the nucleotides encoded within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such combinations, the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such combinations, the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143 or position K147. In some such combinations, the mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution or an M145Y substitution. In some such combinations, the mutation comprises an M145K substitution. In some such combinations, the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution or a W90D substitution. In some such combinations, the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0071] In some such combinations, the IL2RG antagonist is an antigen-binding protein. In some such combinations, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such combinations, the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the same, and wherein the three heavy chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the same. In some such combinations, the three light chain CDRs each comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the same, and the three heavy chain CDRs each comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the same. In some such combinations, the immunoglobulin light chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the same, and the immunoglobulin heavy chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the same. In some such combinations, the immunoglobulin light chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the same, and the immunoglobulin heavy chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the same.

[0072] In some such combinations, the cells are hematopoietic cells. In some such combinations, the cells are lymphocytes or lymphoid progenitor cells. In some such combinations, the cells are T cells. In some such combinations, the cells are tumor-infiltrating lymphocytes (TIL). In some such combinations, the cells are B cells. In some such combinations, the cells are NK cells. In some such combinations, the cells are hematopoietic stem cells, or hematopoietic stem cells and progenitor cells. In some such combinations, the cells are derived from induced pluripotent stem cells. In some such combinations, the cells are derived from hematopoietic stem cells, or hematopoietic stem cells and progenitor cells. In some such combinations, the subject is a mammal or non-human mammal, and the cells are mammalian cells or non-human mammalian cells. In some such combinations, the subject is human, and the cells are human cells. In some such combinations, the cells contain or express a therapeutic molecule. In some such combinations, the cells contain or express an immunoglobulin. In some such combinations, the cells contain a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR). In some such combinations, the cells are autologous. In some such combinations, the cells are allogeneic or syngeneic.

[0073] In some such combinations, the subject has a hematopoietic malignancy, and the combination of agents is used to treat the subject's hematopoietic malignancy. In some such combinations, the subject has cancer. In some such combinations, the cancer is a hematological cancer. In some such combinations, the subject has defective immune cells or a hematopoietic genetic defect. In some such combinations, the hematopoietic genetic defect is sickle cell disease or severe combined immunodeficiency (SCID).

[0074] On the other hand, there are provided isolated cells or cell populations that have been modified to express a first subtype of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second subtype of IL2RG. In some such cells or cell populations, the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable. In some such cells or cell populations, the genomic locus has been edited to express the first subtype of IL2RG in one or more cells. In some such cells or cell populations, the genomic locus is the IL2RG genomic locus. In some such cells or cell populations, the genomic locus is not the IL2RG genomic locus. On the other hand, there are provided isolated cells or cell populations in which the IL2RG genomic locus has been edited to express a first subtype of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second subtype of IL2RG, wherein the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable. In some such cells or cell populations, one or more cells express the first subtype of IL2RG and the second subtype of IL2RG. In some such cells or cell populations, one or more cells express only the first subtype of IL2RG.

[0075] In some such cells or cell populations, the first subtype of IL2RG is a genetically engineered subtype of IL2RG. In some such cells or cell populations, the first subtype of IL2RG is genetically engineered to contain a mutation that provides an altered epitope, optionally, wherein the mutation is an artificial mutation.

[0076] In some such cells or cell populations, one or more cells further comprise an exogenous donor nucleic acid containing an artificial mutation and a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in a genomic locus. In some such cells or cell populations, one or more cells further comprise an exogenous donor nucleic acid containing an artificial mutation and a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in the IL2RG genomic locus. In some such cells or cell populations, the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein; and (ii) a guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that serves as a nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such cells or cell populations, the nuclease reagent comprises a Cas protein and a guide RNA, optionally wherein the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 76-87, or optionally wherein the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 64-75. In some such cells or cell populations, the Cas protein is a Cas9 protein. In some such cells or cell populations, the exogenous donor nucleic acid comprises homology arms. In some such cells or cell populations, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN), optionally wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 88-117.

[0077] In some such cells or cell populations, the altered epitope is in the binding region of an antibody that comprises an immunoglobulin light chain or its variable region, which comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NOs: 12, 14, and 16, and wherein the three heavy chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NOs: 4, 6, and 8.

[0078] In some such cells or cell populations, the mutation comprises a mutation in the nucleotide encoding within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such cells or cell populations, the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such cells or cell populations, the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143 or position K147. In some such cells or cell populations, the mutation comprises a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution or an M145Y substitution. In some such cells or cell populations, the mutation comprises an M145K substitution. In some such cells or cell populations, the mutation comprises a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution or a W90D substitution. In some such cells or cell populations, the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0079] In some such cells or cell populations, the first and second subtypes are functionally indistinguishable but immunologically distinguishable by an IL2RG antagonist. In some such cells or cell populations, the IL2RG antagonist is an antigen-binding protein. In some such cells or cell populations, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such cells or cell populations, the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same. In some such cells or cell populations, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same. In some such cells or cell populations, the immunoglobulin light chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consist essentially of or consist of the same, and the immunoglobulin heavy chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consist essentially of or consist of the same. In some such cells or cell populations, the immunoglobulin light chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 10, consist essentially of or consist of the same, and the immunoglobulin heavy chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 2, consist essentially of or consist of the same.

[0080] In some such cells or cell populations, one or more cells are hematopoietic cells. In some such cells or cell populations, one or more cells are lymphocytes or lymphoid progenitor cells. In some such cells or cell populations, one or more cells are T cells. In some such cells or cell populations, one or more cells are tumor infiltrating lymphocytes (TILs). In some such cells or cell populations, one or more cells are B cells. In some such cells or cell populations, one or more cells are NK cells. In some such cells or cell populations, one or more cells are hematopoietic stem cells, or hematopoietic stem cells and progenitor cells. In some such cells or cell populations, one or more cells are induced pluripotent stem cells. In some such cells or cell populations, one or more cells are mammalian cells or non-human mammalian cells. In some such cells or cell populations, one or more cells are human cells. In some such cells or cell populations, one or more cells contain or express a therapeutic molecule. In some such cells or cell populations, one or more cells contain or express an immunoglobulin. In some such cells or cell populations, one or more cells contain a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR). In some such cells or cell populations, one or more cells are isolated from a subject. In some such cells or cell populations, one or more cells are used to treat a subject having cells expressing a second isoform of IL2RG. In some such cells or cell populations, one or more cells are isolated from the subject.

[0081] In another aspect, a method of preparing any of the above isolated cells or cell populations is provided. In some such methods, the method includes modifying the cells or cell population to express a first isoform of IL2RG. In some such methods, the modification includes editing a genomic locus to express a first isoform of IL2RG. In some such methods, the genomic locus is the IL2RG genomic locus. In some such methods, the genomic locus is not the IL2RG genomic locus. In some such methods, the method includes editing the IL2RG genomic locus to express a first isoform of IL2RG.

[0082] In some such methods, editing comprises introducing into the cell: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in a genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the genomic locus and the exogenous donor nucleic acid inserts into or recombines with the genomic locus to produce an edited cell that expresses a first subtype of IL2RG. In some such methods, editing comprises introducing into the cell: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in the IL2RG genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the IL2RG genomic locus and the exogenous donor nucleic acid inserts into or recombines with the IL2RG genomic locus to produce an edited cell that expresses a first subtype of IL2RG. In some such methods, the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein; and (ii) a guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that is the nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the nuclease reagent comprises a Cas protein and a guide RNA, optionally wherein the DNA targeting fragment comprises a sequence shown in any of SEQ ID NO: 76-87, or optionally wherein the guide RNA target sequence comprises a sequence shown in any of SEQ ID NO: 64-75. In some such methods, the Cas protein is a Cas9 protein. In some such methods, the exogenous donor nucleic acid comprises homology arms. In some such methods, the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN), optionally wherein the ssODN comprises a nucleic acid sequence shown in any of SEQ ID NO: 88-117.

[0083] In another aspect, there is provided a genetically engineered interleukin-2 receptor subunit gamma (IL2RG) protein that contains an artificial mutation to provide an altered epitope. In some such proteins, the genetically engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable but immunologically distinguishable. In another aspect, there is provided a genetically engineered interleukin-2 receptor subunit gamma (IL2RG) protein that contains an artificial mutation to provide an altered epitope, wherein the genetically engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable but immunologically distinguishable. In some such proteins, the altered epitope is in the binding region of an antibody that contains an immunoglobulin light chain or its variable region, which contains three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which contains three heavy chain CDRs, wherein the three light chain CDRs respectively contain the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively contain the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same. In some such proteins, the mutation contains a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus. In some such proteins, the mutation contains a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97. In some such proteins, the artificial mutation contains a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, or position K147. In some such proteins, the artificial mutation contains a mutation or substitution at position M145, optionally wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution. In some such proteins, the artificial mutation contains an M145K substitution. In some such proteins, the mutation contains a mutation or substitution at position W90, optionally wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution. In some such proteins, the mutation contains a mutation or substitution at position M145 and a mutation or substitution at position W90.

[0084] In some such proteins, the engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable, but are immunologically distinguishable by an IL2RG antagonist. In some such proteins, the IL2RG antagonist is an antigen-binding protein. In some such proteins, the antigen-binding protein is an antibody or an antigen-binding fragment thereof. In some such proteins, the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences, and wherein the three heavy chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences. In some such proteins, the three light chain CDRs each comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the sequences, and the three heavy chain CDRs each comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the sequences. In some such proteins, the immunoglobulin light chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence, and the immunoglobulin heavy chain or a variable region thereof comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence. In some such proteins, the immunoglobulin light chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the sequence, and the immunoglobulin heavy chain or a variable region thereof comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the sequence. In some such proteins, the IL2RG protein is a human IL2RG protein.

[0085] In another aspect, provided is a nucleic acid encoding any of the above-described engineered IL2RG proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 A schematic diagram showing the antibody-resistant modified receptor (ARMoR) concept, in which targeted host modulation is combined with donor cell editing to enhance cell-based therapies and transplantation. The concept is a two-layer approach that provides a competitive advantage to transplanted cells: (1) targeted inhibition of host cells (e.g., T cells, NK cells, B cells, lymphoid progenitors, hematological cancers) with an antibody targeting IL2RG; and (2) ex vivo modification of transplanted cells (IL2RG variant - antibody-resistant modified receptor (ARMoR)) to improve survival in the host. The IL2RG variant retains cytokine signaling function but does not bind to anti-IL2RG antibodies.

[0087] Figures 2A - 2B showed that blocking IL2RG to inhibit host T cells created a niche for the persistent engraftment of donor REGN7257-resistant T cells in Il2rg hu / hu mice. Figure 2A showed a schematic of the experimental protocol. REGN7257 or REGN1945 isotype control was injected subcutaneously twice weekly at 25 mg / kg starting on day -25 (indicated by solid arrows). On day 0, 20 x 10 6 donor CD4+ and CD8+ T cells (open arrows) were injected, and bone marrow, spleen, and blood samples were collected on day 24 (open arrows). Figure 2B showed that anti-IL2RG modulation promoted the uptake of "ARMoR’d" donor T cells, which persisted and proliferated within 3 weeks after transfer. At the indicated time points after transfer, blood was collected from mice treated with REGN7257 or isotype control, and the proportion of donor T cells was quantified by flow cytometry (upper row, percentage of total T cells) or counted (lower row, cells / μL blood). Data are represented as mean + / - SEM. Using a two-stage step-up (Benjamini, Krieger, and Yekutieli) multiple comparison test, multiple t-tests were performed between the REGN1945-treated group (n = 5 mice) and the REGN7257-treated group (n = 6 mice) for weekly blood collections, with the false discovery rate (FDR) set at 1%. Statistical significance of the adjusted p-values was recorded as follows: * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001.

[0088] Figures 3A - 3C showed that blocking IL2RG to inhibit host T cells enhanced the MC38-OVA tumor infiltration of antigen-specific, REGN7257-resistant T cells in Il2rg hu / hu mice. Figure 3A showed a schematic of the experimental protocol. REGN7257 or REGN1945 isotype control was injected subcutaneously twice weekly at 25 mg / kg starting on day -25 (indicated by solid arrows). On day 0, MC38 tumor cells expressing ovalbumin (OVA) antigen were implanted (open arrow), and on day 3, 0.1 - 1 x 10 6 donor CD8+ T cells from OT-I mice (open arrows) were injected, and bone marrow, spleen, blood, tumor-draining lymph nodes, and tumor samples were collected on day 24 (open arrowheads). Figures 3B - 3C showed that in REGN7257-modulated Il2rg hu / huIn mice, donor OT-I CD8+ T cells predominated among tumor-infiltrating T cells. Three weeks after implantation, the tumor-infiltrating leukocyte population was quantified by flow cytometry. Figure 3B Representative plots showing quantification of total CD4+ and CD8+ T cells (left column) and OVA-specific T cells identified by pMHC tetramer staining (right column) in mice receiving the indicated treatments. Figure 3C Shows the proportion (upper row) and count (lower row, number of cells / mg tumor) of the indicated cell populations in all mice in the study. Data are represented as mean + / - SEM. The following groups were compared using ordinary one-way ANOVA: REGN1945 treatment, no OT-I transfer (n = 5 mice), REGN1945 treatment, transfer of 1E6 OT-I cells (n = 3 mice), REGN7257 treatment, no OT-I transfer (n = 6 mice), REGN7257 treatment, transfer of 1E5 OT-I cells (n = 3 mice), and REGN7257 treatment, transfer of 1E6 OT-I cells (n = 6 mice). Tukey's multiple comparison test was performed, and the statistical significance of the adjusted p-values was recorded as follows: * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001.

[0089] Figures 4A - 4C Shows that REGN7257 modulation significantly enhanced the infiltration of "ARMoR’d" T cells into the draining lymph nodes of B16.F10.9-OVA tumors. Figure 4A Shows a schematic of the experimental protocol. REGN7257 or REGN1945 isotype control was injected subcutaneously twice weekly at 25 mg / kg starting on day -25 (indicated by solid arrows). B16.F10.9 tumor cells expressing ovalbumin (OVA) antigen were implanted on day 0 (hollow arrow), 1x10 6 donor CD8+ T cells from OT-I mice were injected on day 3 (hollow arrow), and samples were taken on day 24 (hollow arrowhead). Figures 4B - 4C Shows that REGN7257-modulated mice showed enhanced infiltration of tumor-specific OT-I T cells into tumor-draining lymph nodes. Three weeks after implantation, the leukocyte population in tumor-draining lymph nodes (LNs) was quantified by flow cytometry. Figure 4B Representative plots showing quantification of total CD4+ and CD8+ T cells (left column) and OVA-specific T cells identified by pMHC staining (right column) in mice receiving the indicated treatments. Figure 4CShows the percentages (upper row) and counts (lower row, number of cells / LN) of the specified cell populations in all mice in the study. Data are represented as mean + / - SEM. The following groups were compared using ordinary one-way ANOVA: REGN1945 treatment, no OT-I transfer (n = 7 mice), REGN1945 treatment, transfer of 1E6 OT-I cells (n = 7 mice), REGN7257 treatment, no OT-I transfer (n = 5 mice), and REGN7257 treatment, transfer of 1E6 OT-I cells (n = 7 mice). Tukey's multiple comparison test was performed, and the statistical significance of the adjusted p-values was recorded as follows: * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001.

[0090] Figures 5A - 5D Shows that REGN7257 modulation significantly enhanced the ability of adoptively transferred "ARMoR’d" T cells to regress established B16.F10.9-OVA tumors, while enhancing tumor infiltration. Figure 5A Shows a schematic of the experimental protocol. REGN7257 or REGN1945 isotype control was injected subcutaneously twice a week at 25 mg / kg starting on day -25 (indicated by solid arrows). B16.F10.9 tumor cells expressing ovalbumin (OVA) antigen were implanted on day 0 (hollow arrow), and 1 x 10 6 donor CD8+ T cells from OT-I mice were injected on days 13, 18, and 24 (hollow arrows), and then samples were taken for analysis. Figure 5B Shows that REGN7257-modulated mice that received OT-I T cell infusion starting on day 13 after tumor implantation showed enhanced tumor regression, as measured by tumor volume, compared to isotype (REGN1945)-modulated mice. Figure 5C Shows a representative flow cytometry plot (left) and quantitative summary (right) of CD8+ T cell infiltration into tumor-draining lymph nodes (LN). Infiltration of OVA-specific OT-I T cells identified by pMHC tetramer staining was enhanced in REGN7257-modulated mice. Figure 5DShows the quantification of tumor-infiltrating OVA-specific T cells as a proportion of total viable cells isolated from dissected tumors. Infiltration of OVA-specific OT-I T cells identified by pMHC tetramer staining was enhanced in REGN7257-treated mice. Data are represented as mean + / − SEM. The following groups were compared using ordinary one-way ANOVA: REGN1945 treatment, no OT-I transfer (n = 8 mice), REGN1945 treatment, transfer of 1E6 OT-I cells (n = 8 mice), REGN7257 treatment, no OT-I transfer (n = 6 mice), and REGN7257 treatment, transfer of 1E6 OT-I cells (n = 7 mice). Tukey's multiple comparisons test was performed, and statistical significance of adjusted p-values was recorded as follows: * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001.

[0091] Figures 6A - 6E Shows the generation of REGN7257 binding region 1 and variants. Figure 6A Shows the comparison of human and mouse IL2RG sequences in binding region 1 and the generation of mutants. Figure 6B Shows the expression of IL2RG (myc-tagged) variants and quantification of REGN7257 binding. Constructs were transfected into 293T cells, and cells were stained with anti-myc (left column) or REGN7257 (right column). Figure 6C Shows the binding of REGN7257 to YT cells expressing IL2RG variants. YT cells were transduced with lentiviral vectors encoding the indicated variants and stained with REGN7257 at the indicated concentrations on the x-axis. Binding was quantified by flow cytometry (y-axis, gMFI = geometric mean fluorescence intensity). Figures 6D - 6E Shows the screening for additional substitutions at M145. Figure 6D Shows the binding of anti-myc to myc-tagged IL2RG variants with the indicated amino acid substitutions at M145 expressed in 293T cells. Figure 6E Shows the binding of REGN7257 to myc-tagged IL2RG variants with the indicated amino acid substitutions at M145 expressed in 293T cells. Binding was quantified by flow cytometry (y-axis, gMFI = geometric mean fluorescence intensity).

[0092] Figures 7A - 7F Shows that IL2RG variants with mutations in REGN7257 binding region 1 respond to γ-chain cytokines, signal effectively, and escape signal blockade by REGN7257. Figure 7AShows the expression of endogenous IL2RG in YT.STAT5.luc cells engineered with a STAT5-responsive luciferase reporter (top row) compared to YT.STAT5.luc cells in which endogenous IL2R has been knocked out and the indicated IL2RG variants have been re-introduced by lentiviral transduction (remaining rows). Figure 7B Shows luciferase activity in YT.STAT5.luc cells expressing the indicated IL2RG variants in response to titration of human IL2 (triangles) or in the presence of REGN7257 (squares) or isotype (circles) at a constant IL2 concentration (3.3e-11 M). Figure 7C Shows luciferase activity in YT.STAT5.luc cells expressing the indicated IL2RG variants in response to titration of human IL15 (triangles) or in the presence of REGN7257 (squares) or isotype (circles) at a constant IL15 concentration (3.9e-11 M). Figure 7D Shows luciferase activity in YT.STAT5.luc.hIL7Ra cells expressing the indicated IL2RG variants in response to titration of human IL7 (triangles) or in the presence of REGN7257 (squares) or isotype (circles) at a constant IL7 concentration (2.9e-11 M). Figure 7E Shows luciferase activity in YT.STAT5.luc cells expressing the indicated IL2RG variants in response to titration of human IL21 (triangles) or in the presence of REGN7257 (squares) or isotype (circles) at a constant IL21 concentration (3.2e-11 M). Figure 7F Shows luciferase activity in Ramos.STAT3.luc cells expressing the indicated IL2RG variants in response to titration of human IL4 (triangles) or in the presence of REGN7257 (squares) or isotype (circles) at a constant IL4 concentration (3.6e-11 M).

[0093] Figure 8 Shows that REGN7257 inhibits the growth of primary human T cells in vitro. T cells isolated from peripheral blood were activated and grown in the presence of REGN7257 (squares) or isotype (circles) and at low (20 U / mL, left panel) or high (100 U / mL) levels of human IL2.

[0094] Figure 9A-B shows that IL2RG variants with mutations in the REGN7257 binding region 1 support IL2-dependent growth of primary T cells and confer resistance to REGN7257 growth inhibition. T cells isolated from human peripheral blood were activated and transduced with a lentiviral vector co-expressing an IL2RG variant and green fluorescent protein (GFP). Cells were then cultured in the presence of human IL2 and REGN7257 or isotype, and cells expressing the indicated IL2RG variant were quantified over time based on GFP expression. Figure 9A Shown is that REGN7257 treatment (lower panel) increased the proportion of GFP+ cells expressing the REGN7257-resistant variant, but had no such effect on the REGN7257-sensitive variant. In either case, isotype treatment (upper panel) did not alter the proportion of GFP+ cells. Thus, cells expressing an IL2RG variant resistant to REGN7257 escaped growth inhibition and had a growth advantage compared to non-transduced T cells. Figure 9B Shown is a comparison of the IL2-dependent growth kinetics of primary T cells expressing the transduced IL2RG variant (GFP+, squares) compared to non-transduced cells expressing only endogenous IL2RG (GFP-, circles). In the presence of REGN7257, GFP+ cells expressing the REGN7257-resistant variant (lower row) but not the REGN7257-sensitive variant (upper row) showed similar expansion to non-transduced cells. Thus, these cells (cells expressing an IL2RG variant with a mutation disrupting REGN7257 binding in the REGN7257 binding region 1) escaped growth inhibition and supported normal IL2-dependent growth of human T cells.

[0095] Figures 10A - 10C Shown is the screening and optimization of sgRNAs targeting REGN7257 binding region 1 encoded within exon 3 of IL2RG in human T cells. Figure 10A Shown is a schematic of editing exon 3 of the human IL2RG locus. Figure 10B Shown is the editing rate (indel formation) of 12 sgRNAs targeting exon 3. Three sgRNAs (sgRNA 2, 8, and 11) showed the highest indel rates and were further tested separately in the presence of an ssODN donor template. Figure 10C Shown is the editing rate (introduction of the M145K mutation) when sgRNAs 2, 8, and 11 were co-transfected with 10 different ssODN designs (D1-D10), each with a different length, symmetry, and sense / antisense orientation relative to the sgRNA cleavage site.

[0096] Figures 11A - 11CShows the expression and binding to REGN7257 of an IL2RG variant having a mutation in the REGN7257 binding region 2. Figure 11A Shows a comparison of human and mouse IL2RG sequences in binding region 2 (upper panel), and the expression and binding to REGN7257 of the IL2RG (myc-tagged) W90 variant measured by flow cytometry (lower panel). Constructs were transfected into 293T cells and the cells were stained with anti-myc (left column) or REGN7257 (right column). Figure 11B Shows the quantification of the expression of transfected myc-tagged IL2RG variants by flow cytometry (gMFI = geometric mean fluorescence intensity). Figure 11C Shows the quantification of the binding of REGN7257 to transfected IL2RG variants (gMFI = geometric mean fluorescence intensity).

[0097] Figures 12A - 12E Shows that IL2RG variants having a mutation in the REGN7257 binding region 2 respond to γ-chain cytokines, signal effectively, and evade signal blockade by REGN7257. Figure 12A Shows the luciferase activity in YT.STAT5.luc cells expressing the indicated IL2RG variants in response to titration of human IL2 (triangles) or in the presence of constant IL2 concentration (3.3e-11 M) in REGN7257 (squares) or isotype (circles). Figure 12B Shows the luciferase activity in YT.STAT5.luc cells expressing the indicated IL2RG variants in response to titration of human IL15 (triangles) or in the presence of constant IL15 concentration (3.9e-11 M) in REGN7257 (squares) or isotype (circles). Figure 12C Shows the luciferase activity in YT.STAT5.luc.hIL7Ra cells expressing the indicated IL2RG variants in response to titration of human IL7 (triangles) or in the presence of constant IL7 concentration (2.9e-11 M) in REGN7257 (squares) or isotype (circles). Figure 12D Shows the luciferase activity in YT.STAT5.luc cells expressing the indicated IL2RG variants in response to titration of human IL21 (triangles) or in the presence of constant IL21 concentration (3.2e-11 M) in REGN7257 (squares) or isotype (circles). Figure 12E Shows the luciferase activity in Ramos.STAT3.luc cells expressing the indicated IL2RG variants in response to titration of human IL4 (triangles) or in the presence of constant IL4 concentration (3.6e-11 M) in REGN7257 (squares) or isotype (circles).

[0098] Figure 13 Shown is that IL2RG variants with mutations in REGN7257 binding region 2 support IL2-dependent growth of primary T cells and confer resistance to REGN7257 growth inhibition. T cells isolated from human peripheral blood were activated and transduced with a lentiviral vector co-expressing an IL2RG variant and green fluorescent protein (GFP). Cells were then cultured in the presence of human IL2 and REGN7257 or isotype, and cells expressing the indicated IL2RG variant were quantified over time based on GFP expression. The kinetic growth curves of primary T cells (GFP+, squares) expressing the transduced IL2RG variants were compared to non-transduced cells (GFP-, circles) expressing only endogenous IL2RG. In the presence of REGN7257, GFP+ cells expressing the REGN7257-resistant variant (lower line) but not the REGN7257-sensitive variant (upper line) showed amplification similar to that of non-transduced cells. Thus, the cells (cells expressing an IL2RG variant with a mutation disrupting REGN7257 binding in REGN7257 binding region 2) escape growth inhibition and support normal IL2-dependent growth of human T cells.

[0099] Figures 14A - 14C Shown is the screening and optimization of sgRNAs targeting REGN7257 binding region 2 encoded within exon 2 of IL2RG in human T cells. Figure 14A Shown is a schematic of editing exon 2 of the human IL2RG locus. Figure 14B Shown is the editing rate (indel formation) of 18 sgRNAs targeting exon 2. In the presence of an ssODN donor template, two sgRNAs (sgRNA 14 and 15) targeting near the codon encoding W90 and showing high indel rates were further tested. Figure 14C Shown is the editing rate (introducing the W90Q mutation) when sgRNAs 14 and 15 were co-transfected with 10 different ssODN designs (D1-D10), each design having a different length, symmetry, and sense / antisense orientation relative to the sgRNA cleavage site.

[0100] Figures 15A - 15D Shown is the generation of iNK ARMoR by targeted editing of the endogenous IL2RG locus to introduce resistance to REGN725. Figure 15A Shown is the schematic design for generating iNK ARMoR by knocking in ARMoR (IL2RG M145K) in induced pluripotent stem cells (iPSCs) and differentiating the iPSCs ARMoR into iPSC-derived NK (iNK) cells. Figure 15B Shown is iPSC ARMoR having the same as iPSCWT Similar NK differentiation potential. iPSCs were differentiated into iNK cells in vitro, and the proportion of the resulting CD45+CD56+iNK cells was determined by flow cytometry. WT and iPSCs ARMoR cells were differentiated into iNK cells, and the proportion of the resulting CD45+CD56+iNK cells was determined by flow cytometry. Figure 15C Shown is that, as measured by flow cytometry, compared to iNK WT cells, iNK ARMoR cells have reduced REGN7257 binding. Figure 15D Shown is that iNK ARMoR and iNK WT cells bind equivalently to a commercial anti-IL2RG that recognizes an epitope distant from REGN7257.

[0101] Figure 16 Shown is that iNK ARMoR cells evade the inhibition of REGN7257 on IL2- and IL15-dependent growth. Differentiated iNK ARMoR and iNK WT cells were grown with IL2 (left panel) or IL15 (right panel) in the presence of REGN7257, isotype (REGN1945), or no antibody. Cell counts were determined at the indicated time points and reported as normalized to the cell count on day 0 (D0).

[0102] Figures 17A - 17B Shown is that human iNK ARMoR cells implanted into immunodeficient mice are resistant to the inhibition by REGN7257. Figure 17A Shown is a schematic diagram of the experimental protocol. Figure 17B Shown is that in the presence of REGN7257, iNK ARMoR has a competitive advantage in vivo over iNK WT . iNK ARMoR and iNK WT cells were administered to irradiated host mice treated with REGN7257 or isotype control at a 1:1 ratio. At the indicated time points after transfer (transplantation), iNK cells in peripheral blood (proportion of blood leukocytes) were quantified by flow cytometry. A Student t-test was performed, and the statistical significance of the p-value was recorded as follows: **<0.01. Definition

[0103] The terms "protein", "proteinaceous", "polypeptide", and "peptide", which are used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids, as well as chemically or biochemically modified or derivatized amino acids. These terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide that has a specific function or structure.

[0104] Proteins are considered to have an "N-terminus" (amino terminus) and a "C-terminus" (carboxyl terminus). The term "N-terminus" refers to the start of a protein or polypeptide, the terminus of which is an amino acid with a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide), the terminus of which is a free carboxyl group (-COOH).

[0105] The terms "nucleic acid" and "polynucleotide", which are used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or their analogs or modified forms. They include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, unnatural, or derivatized nucleobases.

[0106] Nucleic acids are considered to have a "5'-end" and a "3'-end" because the manner in which mononucleotides react to form oligonucleotides is such that the 5'-phosphate of one mononucleotide pentose ring is joined in one direction by a phosphodiester bond to the 3'-oxygen of its neighboring nucleotide. If the 5'-phosphate of an oligonucleotide is not joined to the 3'-oxygen of a mononucleotide pentose ring, the end of the oligonucleotide is called the "5'-end". If the 3'-oxygen of an oligonucleotide is not joined to the 5'-phosphate of another mononucleotide pentose ring, the end of the oligonucleotide is called the "3'-end". A nucleic acid sequence, even within a larger oligonucleotide, can be considered to have 5' and 3' ends. In linear or circular DNA molecules, discrete elements are referred to as "downstream" or 3' elements "upstream" or 5'.

[0107] The term "expression vector" or "expression construct" or "expression cassette" refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the coding sequence operably linked in a particular host cell or organism. The nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. It is well known that eukaryotic cells utilize promoters, enhancers, and termination and polyadenylation signals, although some elements can be omitted and other elements added without sacrificing necessary expression.

[0108] A "promoter" is a regulatory region of DNA that typically contains a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at an appropriate transcription start site of a specific polynucleotide sequence. Generally, a "promoter" or "promoter sequence" is a DNA regulatory region that can bind RNA polymerase in a cell (e.g., directly or through other promoter-binding proteins or substances) and initiate transcription of a coding sequence. A promoter can be operably linked to other expression control sequences, including enhancer and repressor sequences and / or the polynucleotides of the present invention. Promoters useful for controlling gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062, each of which is incorporated herein by reference in its entirety for all purposes), the SV40 early promoter region (Benoist et al. (1981) Nature 290:304-310, which is incorporated herein by reference in its entirety for all purposes), the promoter contained in the Rous sarcoma virus 3' long terminal repeat (Yamamoto et al. (1980) Cell 22:787-797, which is incorporated herein by reference in its entirety for all purposes), the herpes thymidine kinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, which is incorporated herein by reference in its entirety for all purposes), the regulatory sequences of the metallothionein gene (Brinster et al. (1982) Nature 296:39-42, which is incorporated herein by reference in its entirety for all purposes); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731, which is incorporated herein by reference in its entirety for all purposes) or the tac promoter (DeBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25; see also "Useful Proteins from Recombinant Bacteria" in Scientific American (1980) 242:74-94, each of which is incorporated herein by reference in its entirety for all purposes); and promoter elements from yeast or other fungi such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerate kinase) promoter, or the alkaline phosphatase promoter.

[0109] In some embodiments, a promoter may additionally contain other regions that affect the rate of transcription initiation. A promoter sequence regulates the transcription of an operably linked polynucleotide. A promoter may be active in one or more cell types (such as, but not limited to, eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single-cell stage embryos, differentiated cells, or combinations thereof). For example, a promoter may be a constitutively active promoter, a conditional promoter, an inducible promoter, a time-limited promoter (such as, but not limited to, a developmentally regulated promoter), or a spatially restricted promoter (such as, but not limited to, a cell-specific or tissue-specific promoter).

[0110] "Operably linked" or "operably connected" includes the juxtaposition of two or more components (such as, but not limited to, a promoter and another sequence element) such that the two components function properly and allows the possibility that at least one component can regulate the function exerted on at least one other component. As a non-limiting example, a promoter may be operably linked to a coding sequence if the promoter controls the transcription level of the coding sequence in response to the presence or absence of one or more transcriptional regulators. An operable linkage may include sequences that are adjacent to each other or act in trans (such as, but not limited to, regulatory sequences that can act at a distance to control the transcription of a coding sequence). When, in a cell or other expression system, a polynucleotide encoding a polypeptide is "operably linked" to a promoter or other expression control sequence, the sequence directs RNA polymerase-mediated transcription of the coding sequence into RNA (preferably mRNA), which can then be spliced (if it contains introns) and optionally translated into the protein encoded by the coding sequence.

[0111] As used with respect to proteins, nucleic acids, and cells, the term "isolated" includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or organismal components that are normally present in situ, up to and including substantially pure preparations of proteins, nucleic acids, or cells. In some embodiments, the term "isolated" may include proteins and nucleic acids that do not have a naturally occurring counterpart, or proteins or nucleic acids that have been chemically synthesized and are thus substantially free from contamination by other proteins or nucleic acids. The term "isolated" may include proteins, nucleic acids, or cells that are separated or purified from most other cellular components or organismal components with which they are naturally associated (e.g., but not limited to other cellular proteins, nucleic acids, or cells or extracellular components). An "isolated" antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof), polypeptide, polynucleotide, and vector are at least partially free of other biomolecules in the cell or cell culture in which they are produced. Such biomolecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth medium. An isolated antigen-binding protein may also be at least partially free of expression system components, such as biomolecules from a host cell or its growth medium. In general, the term "isolated" does not mean the complete absence of such biomolecules (e.g., small amounts or trace amounts of impurities may remain), nor does it mean the absence of water, buffer, salts, or components of a pharmaceutical formulation containing the antigen-binding protein (e.g., an antibody or an antigen-binding fragment).

[0112] "Codon optimization" takes advantage of the degeneracy of the genetic code, as demonstrated by the multiplicity of three-base pair codon combinations that specify an amino acid, and generally involves modifying a nucleic acid sequence to enhance expression in a particular host cell by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. As a non-limiting example, a nucleic acid encoding a protein can be modified to substitute codons that have a higher usage frequency in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, e.g., in the "Codon Usage Database". These tables can be adjusted in a variety of ways. See Nakamura et al. (2000) Nucleic Acids Res. 28(1):292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (e.g., see Gene Forge).

[0113] The term "locus" refers to the specific location of a gene (or significant sequence), DNA sequence, polypeptide coding sequence, or position on a chromosomal DNA of an organism. As a non-limiting example, the "IL2RG locus" can refer to the specific location of the IL2RG gene, IL2RG DNA sequence, IL2RG protein coding sequence, or the position of IL2RG on the chromosomal DNA of an organism, which has been identified as the location where the sequence is located. The "IL2RG locus" can contain regulatory elements of IL2RG, including, as non-limiting examples, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0114] The term "gene" refers to a DNA sequence in a chromosome that, if occurring naturally, may contain at least one coding region and at least one non-coding region. A DNA sequence in a chromosome that encodes a product (such as, but not limited to, an RNA product and / or a polypeptide product) may include coding regions interrupted by non-coding introns and sequences adjacent to the coding regions at the 5' and 3' ends, such that the gene corresponds to a full-length mRNA (including 5' and 3' untranslated sequences). In addition, there may be other non-coding sequences in a gene, including regulatory sequences (such as, but not limited to, promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulator sequences, and matrix attachment regions. These sequences may be close to the coding region of the gene (such as, but not limited to, within 10 kb) or located at distant sites, and they can affect the transcription and translation levels or rates of the gene.

[0115] The term "allele" refers to variant forms of a gene. Some genes have multiple different forms that are located at the same position on a chromosome or genetic locus. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. If there are two identical alleles at a specific locus, the genotype is described as homozygous, and if the two alleles are different, it is described as heterozygous.

[0116] The term "wild type" includes entities having a structure (such as, but not limited to, a nucleotide sequence or an amino acid sequence) found in a normal (as opposed to mutant, diseased, altered, etc.) state or background. Wild-type genes and polypeptides typically exist in multiple different forms (such as alleles).

[0117] The term "variant" refers to a nucleotide sequence that is different from the most prevalent sequence in a population (such as, but not limited to, differing by one nucleotide) or a protein sequence that is different from the most prevalent sequence in a population (such as, but not limited to, differing by one amino acid).

[0118] When referring to a protein, the term "fragment" means a protein that is shorter or has fewer amino acids than the full-length protein. When referring to a nucleic acid, the term "fragment" means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. Non-limiting examples of protein fragments can include N-terminal fragments (i.e., removing a portion of the C-terminus of the protein), C-terminal fragments (i.e., removing a portion of the N-terminus of the protein), or internal fragments (i.e., removing a portion of the internal part of the protein).

[0119] In the context of two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to residues that are the same in the two sequences when the sequences are aligned maximally for correspondence over a specified comparison window. When percent sequence identity is used in reference to a protein, the positions of non-identical residues are often different because of conservative amino acid substitutions, where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., but not limited to, charge or hydrophobicity), and thus the functional properties of the molecule are not changed. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Methods for making this adjustment are well known. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percent sequence identity. Thus, as a non-limiting example, when identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. The scoring of conservative substitutions is implemented, for example, in the program PC / GENE (Intelligenetics, Mountain View, California).

[0120] "Percent sequence identity" includes the value determined by comparing two optimally aligned sequences over a comparison window (the greatest number of residue matches), where the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (excluding additions or deletions) for optimal alignment of the two sequences. The percent is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and then multiplying the result by 100 to yield the percent sequence identity. Unless otherwise stated (e.g., shorter sequences include linked heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0121] Unless otherwise indicated, sequence identity / similarity values include those obtained using GAP Version 10 with the following parameters: the percent identity and percent similarity of nucleotide sequences obtained using GAP Weight 50 and Length Weight 3 and the nwsgapdna.cmp scoring matrix; the percent identity and percent similarity of amino acid sequences obtained using GAP Weight 8 and Length Weight 2 and the BLOSUM62 scoring matrix; or values obtained using any equivalent program thereof. "Equivalent program" includes any sequence comparison program that, for any two sequences being discussed, generates an alignment having the same nucleotide or amino acid residue matches and the same percent sequence identity as the corresponding alignment generated by GAP Version 10.

[0122] The term "in vitro" includes an artificial environment and processes or reactions occurring in an artificial environment (e.g., but not limited to, test tubes or isolated cells or cell lines). The term "in vivo" includes a natural environment (e.g., but not limited to, an organism or body or cells or tissues within an organism or body) and processes or reactions occurring in a natural environment. The term "ex vivo" includes cells removed from an individual and processes or reactions occurring within those cells.

[0123] "Optional" or "optionally" means that the subsequent described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0124] Specification of a value range includes all integers within that range or defining that range, as well as all sub-ranges defined by the integers within that range.

[0125] Unless the context clearly dictates otherwise, the term "about" includes ±5 of the stated value.

[0126] The term "and / or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0127] The term "or" means any one member of a particular list.

[0128] Unless the context clearly dictates otherwise, the singular forms of the articles "a", "an", and "the" include plural references. For example, the term "protein" or "at least one protein" can include multiple proteins, including mixtures thereof.

[0129] Statistical significance means p ≤ 0.05. Detailed Description I. Overview

[0130] Immune cell therapy holds great promise for many human diseases. One of the oldest examples is bone marrow transplantation, in which the entire immune system of the recipient can be replaced with an autologous or allogeneic bone marrow graft. This procedure allows correction of congenital hematopoietic defects and also allows for reconstitution of the immune system after treatment to eradicate hematological malignancies. More recent examples include immune cells engineered with antigen receptors to target tumors (such as CAR-T, eTCR, CAR-NK, and CAR macrophages). In all of these cases, patients must receive a "conditioning" regimen prior to cell transplantation, which can "make room" in the host immune niche to support engraftment of donor cells and, in some cases, suppress the host anti-graft immune response that can lead to graft rejection.

[0131] The intensity of the conditioning regimen ranges from partial myeloablation to full myeloablation, the latter being necessary when pathogenic host immune cells must be completely eradicated (e.g., for hematological malignancies). In any case, the current standard of care for host conditioning has significant drawbacks. First, the conditioning agents are toxins (such as DNA-damaging agents) that are not specific to the desired target cells and thus pose harmful and even life-threatening risks to the patient. In addition, the conditioning agents are as toxic to donor cells as they are to the host and thus must be discontinued prior to transplantation to avoid inhibition of life-saving cell therapies. These challenges generally limit the application of cell therapies to dire situations where there are no treatment alternatives.

[0132] Lymphoid inhibitors have many potential applications in host conditioning for transplantation and adoptive cell therapy: (1) preventing rejection of allogeneic grafts (such as bone marrow transplantation; gene-corrected cell therapy) through T- and NK-cell inhibition; (2) non-genotoxic clearance of the immune niche space for engineered cell therapies (such as CAR-T, TCR-T, Treg, NK, B cells, progenitors); (3) elimination of endogenous cytokine "sinks" to make essential factors more accessible to graft cells; and (4) immunosuppression after transplantation, which is milder and less toxic than standard-of-care agents.

[0133] One obstacle to using lymphoid inhibitors as conditioning therapies is the sensitivity of transplanted cells to their action in addition to the target host cells. This article provides a strategy for addressing these challenges: (1) developing targeted conditioning regimens that utilize, for example, antibodies that specifically target the desired host cells as single therapies, combinations, bispecific antibodies, antibody-drug conjugates (ADCs), or scFv-engineered CAR-Ts, and (2) modifying donor cells to render them resistant to, for example, these antibody-based conditioning agents. Collectively, this encompasses the antibody-resistant modified receptor (ARMoR) concept. The basic idea is to make minimal alterations to immune cell receptors in transplanted donor cells to eliminate binding of inhibitory antibodies. A schematic of this strategy is shown in Figure 1As shown, a non-limiting example of the use of an engineered interleukin 2 receptor subunit gamma (IL2RG) variant that is resistant to blockade of cytokine signaling necessary for IL2RG-mediated lymphocyte function. The goal is to introduce a fully functional form of the receptor into a cell therapy product that is not recognized by the modulator. This can be achieved by altering the antibody recognition site to eliminate binding while retaining receptor function. Thus, the host cells remain sensitive to the modulator, but the engineered transplanted cells are resistant, and thus gain a competitive advantage in re-populating the host. The basic objective is to provide a competitive advantage for transplanted cell therapies in host patients by imposing a selective pressure specifically targeting the host cells while avoiding donor-derived cell therapies.

[0134] Methods are provided for improving engraftment of donor cells in a subject. Such methods can include providing donor cells that express (e.g., have been modified to express) a first subtype of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), administering the donor cells to the subject, and then selectively depleting host cells in the subject based on the expression of a second subtype of the target protein, thereby improving engraftment of the donor cells in the subject. For example, such methods can include providing donor cells that express (e.g., have been modified to express) a first subtype of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), administering the donor cells to the subject, and then selectively inhibiting host cells in the subject based on the expression of a second subtype of the target protein, thereby improving engraftment of the donor cells in the subject. Alternatively, such methods can include providing donor cells that express (e.g., have been modified to express) a first subtype of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), administering the donor cells to the subject, and then selectively ablating host cells in the subject based on the expression of a second subtype of the target protein, thereby improving engraftment of the donor cells in the subject. The donor cells can express only the first subtype, or they can express the first and second subtypes of the target protein. The first and second subtypes of the target protein are functionally indistinguishable but immunologically distinguishable. In some embodiments, the target protein is a protein expressed on the cell surface of hematopoietic cells (e.g., lymphocytes). In some embodiments, the target protein is a receptor, such as a cytokine receptor or a chemokine receptor (e.g., in some embodiments, the receptor is a cytokine receptor). In some embodiments, the target protein is a subunit of a cytokine receptor interleukin-2 (IL-2) receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor, or IL-21 receptor. For example, the target protein can be IL2RG. In some embodiments, the selective inhibition of host cells can be based on their expression of only the second subtype of the target protein and their lack of expression of the first subtype of the target protein. Alternatively, the selective inhibition of host cells can be based on their expression of the second subtype, regardless of their expression of the first subtype of the target protein. The selective inhibition of host cells does not include ablating (i.e., killing) host cells by an extracellular mechanism (e.g., by an active killing mechanism). The selective inhibition of host cells does not include ablating (i.e., killing) host cells by an active killing mechanism.An active killing mechanism refers to a reagent directly killing host cells through a cytotoxic mechanism (e.g., antibody-drug conjugate (ADC), antibody-radioactive conjugate (ARC), CAR-T, or other engineered cytotoxicity), or recruiting host cell cytotoxic effector mechanisms (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP)), rather than blocking cell functions (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion; e.g., like selective inhibition) without engaging exogenous cytotoxic effectors. These host cell cytotoxic effector mechanisms are well-known. See, e.g., Yu et al. (2020) J. Hematol. Oncol. 13(1):45 and Gogesch et al. (2021) Int. J. Mol. Sci. 22(16):8947, which are each incorporated herein by reference in their entireties for all purposes. As a novel regulatory strategy, selectively inhibiting host cells without cytotoxic ablation has the potential to improve the safety and efficacy of cell therapies and transplantation therapies. Non-ablative regulation can avoid the adverse and harmful effects of ablative agents, including direct killing of non-target cells expressing the drug target antigen (e.g., non-hematopoietic cells), indirect toxicity to target-adjacent tissues, and long-term immunosuppression after transplantation. Selectively blocking or inhibiting essential host cell factors can provide a favorable competition for restricted host factors (e.g., cytokines, chemokines) and immune niche space, thereby enhancing the expansion, persistence, and trafficking of resistant donor cells without using stimulatory and potentially toxic ablative agents. See, e.g., . Figures 2A - 5D . In some embodiments, the selective inhibition of host cells can include: (1) blocking the growth of host cells to provide a competitive growth advantage for donor cells; (2) blocking the localization or trafficking of host cells to provide a competitive homing advantage for donor cells; (3) blocking the cell-cell interaction or adhesion of host cells to provide a competitive tissue infiltration advantage for donor cells; or (4) blocking immune cell activation in host cells to provide a competitive advantage for donor cells. For example, in some embodiments, the selective inhibition of host cells includes blocking growth (i.e., proliferation) and / or blocking immune cell activation. For example, in some embodiments, the selective inhibition of host cells includes blocking growth (i.e., proliferation) and blocking immune cell activation. For example, in some embodiments, the selective inhibition of host cells includes blocking growth (i.e., proliferation). For example, in some embodiments, the selective inhibition includes blocking immune cell activation. Also provided is a combination for administration to a subject in need thereof, wherein the combination comprises (1) a population of donor cells expressing (e.g., modified to express) a first subtype of a target protein (e.g., IL2RG), and (2) a reagent (e.g., an antagonist, e.g., an antigen-binding protein) that specifically binds a second subtype of the target protein but does not specifically bind the first subtype of the target protein.

[0135] Methods are provided for selectively depleting unedited cells in vivo in their subjects. Methods are provided for selectively depleting unedited cells in vivo in their subjects and reconstituting with edited cells. These methods can include providing cells edited to express a first isoform of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), administering the edited cells to a subject, and then selectively depleting unedited cells in the subject based on the expression of a second isoform of the target protein. These methods can include providing cells edited to express a first isoform of a target protein (e.g., interleukin-2 receptor subunit gamma (IL2RG)), the first isoform being functionally indistinguishable but immunologically distinguishable from a second isoform of the target protein, administering the edited cells to a subject, and then selectively depleting unedited cells in the subject based on the expression of the second isoform of the target protein. For example, selective depletion of unedited cells can be based on their expressing only the second isoform of the target protein and their lacking expression of the first isoform of the target protein. Alternatively, they can be depleted based on their expressing the second isoform regardless of their expression of the first isoform of the target protein. The first and second isoforms of the target protein are functionally indistinguishable but immunologically distinguishable. The edited cells can express only the first isoform, or they can express the first and second isoforms of the target protein. Combinations for administration to a subject in need are also provided, wherein the combination comprises (1) a population of cells edited to express a first isoform of a target protein (e.g., IL2RG), and (2) a reagent (e.g., an antagonist, e.g., an anti-IL2RG antigen-binding protein) that specifically binds the second isoform of the target protein but does not specifically bind the first isoform of the target protein.

[0136] Isolated cells or populations of cells are also provided that are modified to express a first isoform of a target protein that is different from a second isoform of the target protein. The cells can express only the first isoform, or they can express the first and second isoforms. In some embodiments, the target protein is a protein expressed on the cell surface of hematopoietic cells (e.g., lymphocytes). In some embodiments, the target protein is a receptor, such as a cytokine receptor or a chemokine receptor (e.g., in some embodiments, the receptor is a cytokine receptor). In some embodiments, the target protein is a subunit of a cytokine receptor interleukin-2 (IL-2) receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor, or IL-21 receptor. For example, the target protein can be IL2RG. The first and second isoforms are functionally indistinguishable but immunologically distinguishable. The cells can express only the first isoform, or they can express the first and second isoforms. Isolated cells or populations of cells are also provided wherein a genomic locus has been edited to express a first isoform of a target protein that is different from a second isoform. Methods for preparing these cells are also provided.

[0137] Also provided are isolated cells or cell populations of a first isoform of IL2RG that have been edited (i.e., modified) to express a second isoform different from IL2RG. The first and second isoforms of IL2RG are functionally indistinguishable but immunologically distinguishable. The edited cells can express only the first isoform, or they can express both the first and second isoforms of IL2RG. Also provided are isolated cells or cell populations in which the genomic locus has been edited to express a first isoform of IL2RG different from a second isoform of IL2RG. The first and second isoforms of IL2RG are functionally indistinguishable but immunologically distinguishable. The edited cells can express only the first isoform, or they can express both the first and second isoforms of IL2RG. Also provided are isolated cells or cell populations in which the IL2RG genomic locus has been edited to express a first isoform of IL2RG different from a second isoform of IL2RG, wherein the first and second isoforms are functionally indistinguishable but immunologically distinguishable. Also provided are methods of preparing these cells, and engineered IL2RG proteins and nucleic acids encoding the engineered IL2R proteins are also provided.

[0138] In some embodiments, the cells (e.g., donor cells or edited cells) in the compositions and methods contain or express a therapeutic molecule, such as a therapeutic protein or enzyme, an immunoglobulin (e.g., an antibody or an antigen-binding fragment thereof), a chimeric antigen receptor (CAR) (e.g., CAR-T cells, CAR-NK cells), or an exogenous T cell receptor (TCR). In some embodiments, the therapeutic molecule, immunoglobulin, CAR, or exogenous TCR does not target a target protein (e.g., IL2RG). For example, the donor cells or edited cells can be engineered to express a therapeutic molecule that has therapeutic activity against any disease, such as any type of cancer (e.g., regardless of whether the target protein is associated with the disease or cancer), including diseases or cancers that are unrelated to the target protein (e.g., the target protein discussed above is not targeted for treating the disease or cancer, but the compositions and methods disclosed herein can provide a competitive advantage for cells that contain or express the therapeutic molecule). For example, the disease or cancer can be a disease or cancer that is unrelated to the target protein (e.g., the target protein does not cause the disease or cancer, and / or the expression of the target protein is not associated with the disease or cancer). In some such embodiments, the therapeutic molecule can target diseased cells and / or antigens expressed on diseased cells (e.g., tumor-associated antigens). II. Methods for Improving Donor Cell Engraftment in a Subject

[0139] In some embodiments of the present invention, methods are provided for improving engraftment of donor cells in a subject. These methods can include providing donor cells that express (e.g., have been modified to express) a first subtype of a target protein that is different from a second subtype of the target protein, wherein the second subtype is expressed in host cells of the subject. The target protein can be, for example, a protein expressed on the cell surface of hematopoietic cells. The first and second subtypes of the target protein are functionally indistinguishable but immunologically distinguishable. In some embodiments, the donor cells express only the first subtype of the target protein. In other embodiments, the donor cells express both the first and second subtypes of the target protein. These methods can include providing donor cells in which the target genomic locus has been edited to express the first subtype of the target protein. The donor cells can then be administered to the subject, and host cells in the subject can be selectively depleted based on their expression of the second subtype of the target protein, thereby improving engraftment of the donor cells in the subject. For example, the selective depletion of host cells can be based on their expression of only the second subtype of the target protein and their lack of expression of the first subtype of the target protein. Alternatively, the selective depletion of host cells can be based on their expression of the second subtype regardless of their expression of the first subtype of the target protein. For example, host cells in the subject can be selectively inhibited based on their expression of the second subtype of the target protein, thereby improving engraftment of the donor cells in the subject. For example, the selective inhibition of host cells can be based on their expression of only the second subtype of the target protein and their lack of expression of the first subtype of the target protein. Alternatively, the selective inhibition of host cells can be based on their expression of the second subtype regardless of their expression of the first subtype of the target protein. Alternatively, host cells in the subject can be selectively ablated based on their expression of the second subtype of the target protein, thereby improving engraftment of the donor cells in the subject. For example, the selective ablation of host cells can be based on their expression of only the second subtype of the target protein and their lack of expression of the first subtype of the target protein. Alternatively, the selective depletion of host cells can be based on their expression of the second subtype regardless of their expression of the first subtype of the target protein.

[0140] In some embodiments of the present invention, methods are provided for selectively depleting unedited cells in a subject. In some embodiments of the present invention, methods are provided for selectively depleting unedited cells in a subject and re-populating with edited cells. These methods can include providing edited cells that have been modified to express a first isoform of a target protein that is different from a second isoform of the target protein, wherein the second isoform is expressed in unedited cells of the subject. The first and second isoforms of the target protein are functionally indistinguishable but immunologically distinguishable. In some embodiments, the edited cells express only the first isoform of the target protein. In other embodiments, the edited cells express the first and second isoforms of the target protein. These methods can include providing edited cells in which a target genomic locus has been edited to express a first isoform of a target protein that is different from a second isoform of the target protein, wherein the second isoform is expressed in unedited cells of the subject. The first and second isoforms of the target protein are functionally indistinguishable but immunologically distinguishable. In some embodiments, the edited cells express only the first isoform of the target protein. In other embodiments, the edited cells express the first and second isoforms of the target protein. These methods can include providing edited cells in which a target genomic locus has been edited to express a first isoform of a target protein that is different from a second isoform of the target protein, wherein the first and second isoforms are functionally indistinguishable but immunologically distinguishable, and wherein the second isoform is expressed in unedited cells of the subject. The edited cells can then be administered to the subject, and the unedited cells in the subject can be selectively depleted based on their expression of the second isoform of the target protein. For example, the selective depletion of unedited cells can be based on their expression of only the second isoform of the target protein and their lack of expression of the first isoform of the target protein. Alternatively, they can be depleted based on their expression of the second isoform regardless of their expression of the first isoform of the target protein.

[0141] The donor cell or edited cell can be any suitable cell. Similarly, the host cell or unedited cell can be any suitable cell. In some embodiments, the cell is a hematopoietic cell. The term hematopoietic cell refers to a cell derived from a hematopoietic stem cell or hematopoietic progenitor cell and / or derived from the erythroid, lymphoid, or myeloid lineage. In some embodiments, the cell is an immune cell. The term immune cell refers to any cell derived from a hematopoietic stem cell that plays a role in the immune response. Immune cells include, but are not limited to, lymphocytes such as T cells and B cells, antigen-presenting cells (APCs), dendritic cells, monocytes, macrophages, natural killer (NK) cells, mast cells, basophils, eosinophils, or neutrophils, and any progenitor cells of these cells. In some embodiments, the cell is a lymphocyte or lymphoid progenitor cell. In some embodiments, the cell is a T cell (e.g., CD4+ T cell, CD8+ T cell, memory T cell, regulatory T cell, γδ T cell, mucosal-associated invariant T cell (MAIT), tumor-infiltrating lymphocyte (TIL), or any combination thereof). In some embodiments, the cell is a TIL. In some embodiments, the cell is a B cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is an innate lymphocyte. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is a hematopoietic stem cell (HSC), or hematopoietic stem and progenitor cells (HSPC), or their progeny. HSCs are capable of giving rise to myeloid and lymphoid progenitor cells, which in turn give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, red blood cells, platelets, etc.) and lymphocytes (e.g., T cells, B cells, NK cells). In some embodiments, the cell is derived from induced pluripotent stem cells (e.g., NK cells derived from induced pluripotent stem cells). In some embodiments, the cell is derived from HSCs or HSPCs.

[0142] In some embodiments, the cells (e.g., donor cells or edited cells) contain genetic modifications (insertion of a transgene, correction of a mutation, gene deletion or inactivation (e.g., insertion of a premature stop codon or insertion of a regulatory repressor sequence), or a change in an epigenetic modification important for gene expression) to correct or counteract a disease-related gene defect present in a subject. In some embodiments, the cells (e.g., donor cells or edited cells) contain a transgene. In some embodiments, the cells (e.g., donor cells or edited cells) contain or express a therapeutic molecule, such as a therapeutic protein or enzyme, an immunoglobulin (e.g., an antibody or an antigen-binding fragment thereof), a chimeric antigen receptor (CAR) (e.g., CAR-T cells, CAR-NK cells), or an exogenous T cell receptor (TCR). In some embodiments, the cells (e.g., donor cells or edited cells) contain a bicistronic nucleic acid construct encoding a therapeutic molecule and a first isoform of a target protein. For examples of bicistronic constructs expressing a CAR and another molecule, see, e.g., Yeku et al. (2017) Sci. Rep. 7(1):10541 and Rafiq et al. (2018) Nat. Biotechnol. 36(9):847-856, each of which is incorporated herein by reference in its entirety for all purposes. For example, the bicistronic construct can encode a therapeutic protein (e.g., a CAR) and a first isoform (e.g., a modified isoform) of a target protein (e.g., IL2RG). In one embodiment, the bicistronic construct encodes a therapeutic protein (e.g., a CAR) and a modified isoform of IL2RG. In some embodiments, the therapeutic molecule, immunoglobulin, CAR, or exogenous TCR does not target the target protein (e.g., IL2RG). In some embodiments, the cells (e.g., donor cells or edited cells) contain or express an immunoglobulin, CAR, or exogenous TCR. In some embodiments, the cells (e.g., donor cells or edited cells) contain a CAR or an exogenous TCR. For example, donor cells or edited cells can be engineered to express a therapeutic molecule having therapeutic activity against any disease, such as any type of cancer (e.g., regardless of whether the target protein is associated with the disease or cancer), including diseases or cancers unrelated to the target protein (e.g., the target protein discussed above is not targeted for treating the disease or cancer, but the compositions and methods disclosed herein can provide a competitive advantage for cells containing or expressing the therapeutic molecule). In some embodiments, the therapeutic molecule targets diseased cells and / or an antigen expressed on diseased cells (e.g., a tumor-associated antigen). For example, the disease or cancer can be a disease or cancer unrelated to the target protein (e.g., the target protein does not cause the disease or cancer, and / or the expression of the target protein is not associated with the disease or cancer). Exemplary types of cancers and tumors that can be treated are described elsewhere herein.

[0143] In some embodiments, the donor cell is autologous (i.e., from the subject). In some embodiments, the donor cell is allogeneic (i.e., not from the subject) or syngeneic (i.e., genetically identical, or sufficiently identical and immunocompatible to allow transplantation). In some embodiments, the cell is a mammalian cell or a non-human mammalian cell (e.g., a mouse or rat cell or a non-human primate cell) (e.g., the subject is a mammal or a non-human mammal, and the donor cell is a mammalian cell or a non-human mammalian cell). In some embodiments, the cell is a human cell (e.g., the subject is human, and the donor cell is a human cell).

[0144] Any suitable target protein can be used. In some embodiments, the target protein is a cell surface protein, such as a receptor. For example, the target protein can be a cell surface protein (such as a receptor, such as a cytokine receptor or chemokine receptor) expressed on the cell surface of hematopoietic cells (such as lymphocytes). In some embodiments, the cell surface protein is selected from CD1a, CD1b, CD1c, CD1e, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDwl2, CD13, CD14, CD15, CD15u, CD15s, CD15su, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85d, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a,CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158e, CD158i, CD158k, CD159a, CD159c, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167a, CD167b, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198 / CDw198, CD199 / CDw199, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210 / CD210A, CD210B / CDw210b, CD212, CD213a1, CD213a2, CD215, CD217a, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240DCE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269(BCMA), CD270, CD271, CD272, CD273, CD274CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, ACKR2, ACKR4, CCR10, CCRL2, CNTFR, CX3CR1, CXCR7, CXCR8, EDA2R, EDAR, EPOR, FLT1, FLT4, GHR, GPR75, IFNAR1, IFNAR2, IFNGR2, IL11RA, IL12RB2, IL17RB, IL17RC, IL17RD, IL17RE, IL1RAP, IL1RAPL, IL1RL1, IL1RL2, IL20RA, IL20RB, IL22RA1, IL22RA2, IL23R, IL27RA, IL28RA, IL31RA, IL3RB, OSMR, PRLR, RELL, RELT, SIGIRR, TNFRSF11B, TNFRSF19, TNFRSF22, TNFRSF23, TNFRSF25, TNFRSF26, TNFRSF3, TNFRSF6B, TSLPR, XCR1, Immunoglobulin light chain (λ or κ), HLA protein (HLA refers to "human leukocyte antigen" and includes HLA-A, HLA.B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP,HLA-DQ and HLA-DR), and β2-microglobulin. In some embodiments, the cell surface protein is selected from CD1a, CD1b, CD1c, CD1e, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDwl2, CD13, CD14, CD15, CD15u, CD15s, CD15su, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85d, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158e, CD158i, CD158k, CD159a, CD159c, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167a, CD167b, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198, CD199, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217a, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240DCE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD266, CD267, CD268, CD269(BCMA), CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298,CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, immunoglobulin light chain (λ or κ), HLA protein (HLA refers to "human leukocyte antigen" and includes HLA-A, HLA.B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP, HLA-DQ and HLA-DR), and β2-microglobulin.

[0145] In some embodiments, the target protein can be a lineage-specific cell surface protein. In some embodiments, the target protein can be selected from CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLECLi); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlep(1-1)Cer); TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn-Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; prostate acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor I receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (Prosome, Macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogenic fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA);O-Acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor family C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycosphingolipid (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta-3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostaglandin; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen recognized by T cells 1 (MelaA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma cell apoptosis inhibitor (ML-AP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or brother of the regulator of imprinted sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1);Lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products-1 (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); leguminous plants; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR 1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2), lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0146] In some embodiments, the target protein is a protein expressed on the cell surface of hematopoietic cells. In some embodiments, the target protein is a receptor expressed on the cell surface of hematopoietic cells. For example, in some embodiments, the target protein may be a receptor expressed on the cell surface of hematopoietic cells, which is selected from ACKR2, ACKR4, CCR10, CCRL2, CD25, CD27, CD30, CD40, CD95, CD110, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD132, CD134, CD135, CD136, CD137, CD140a, CD140b, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198 / CDw198, CD199 / CDw199, CD202b, CD210A, CD210B, CD212, CD213A1, CD213A2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD234, CD246, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD295, CD309, CD331, CD332, CD333, CD334, CD357, CD358, CD360, CNTFR, CX3CR1, CXCR7, CXCR8, EDA2R, EDAR, EPOR, FLT1, FLT4, GHR, GPR75, IFNAR1, IFNAR2, IFNGR2, IL11RA, IL12RB2, IL17RB, IL17RC, IL17RD, IL17RE, IL1RAP, IL1RAPL, IL1RL1, IL1RL2, IL20RA, IL20RB, IL22RA1, IL22RA2, IL23R, IL27RA, IL28RA, IL31RA, IL3RB, OSMR, PRLR, RELL, RELT, SIGIRR, TNFRSF11B, TNFRSF19, TNFRSF22, TNFRSF23, TNFRSF25, TNFRSF26, TNFRSF3, TNFRSF6B, TSLPR, and XCR1.

[0147] In some embodiments, the target protein is a cytokine receptor or chemokine receptor (e.g., expressed on the cell surface of hematopoietic cells). For example, in some embodiments, the target protein can be a cytokine receptor or chemokine receptor expressed on the cell surface of hematopoietic cells, which are selected from CD25, CD30, CD95, CD110, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD132, CD135, CD136, CD140a, CD140b, CD202b, CD210A, CD210B, CD212, CD213A1, CD213A2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD246, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD295, CD309, CD331, CD332, CD333, CD334, CD360, EPOR, FLT1, FLT4, IL11RA, IL12RB2, IL17RB, IL17RC, IL17RD, IL17RE, IL1RAP, IL1RAPL, IL1RL1, IL1RL2, IL20RA, IL20RB, IL22RA1, IL22RA2, IL23R, IL27RA, IL28RA, IL31RA, IL3RB, OSMR, RELT, SIGIRR, TNFRSF25, TNFRSF3, TNFRSF6B, TSLPR, ACKR2, CCRL2, CCR10, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198 / CDw198, CD199 / CDw199, CD234, CXCR7, CXCR8, CX3CR1, GPR75 and XCR1.

[0148] In some embodiments, the target protein is a cytokine receptor (e.g., expressed on the cell surface of hematopoietic cells). For example, in some embodiments, the target protein can be a cytokine receptor expressed on the cell surface of hematopoietic cells, which is selected from CD25, CD30, CD95, CD110, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD132, CD135, CD136, CD140a, CD140b, CD202b, CD210A, CD210B, CD212, CD213A1, CD213A2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD246, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD295, CD309, CD331, CD332, CD333, CD334, CD360, EPOR, FLT1, FLT4, IL11RA, IL12RB2, IL17RB, IL17RC, IL17RD, IL17RE, IL1RAP, IL1RAPL, IL1RL1, IL1RL2, IL20RA, IL20RB, IL22RA1, IL22RA2, IL23R, IL27RA, IL28RA, IL31RA, IL3RB, OSMR, RELT, SIGIRR, TNFRSF25, TNFRSF3, TNFRSF6B, and TSLPR.

[0149] In some embodiments, the target protein is a chemokine receptor (e.g., expressed on the cell surface of hematopoietic cells). For example, in some embodiments, the target protein can be a chemokine receptor expressed on the cell surface of hematopoietic cells, which is selected from ACKR2, CCRL2, CCR10, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198 / CDw198, CD199 / CDw199, CD234, CXCR7, CXCR8, CX3CR1, GPR75, and XCR1.

[0150] In some embodiments, the target protein is a cytokine receptor or chemokine receptor expressed on the cell surface of lymphocytes. For example, in some embodiments, the target protein may be selected from CD25, CD30, CD95, CD117, CD118, CD120a, CD120b, CD121a, CD121b, CD122, CD124, CD125, CD126, CD127, CD129, CD130, CD132, CD210A, CD210B, CD212, CD215, CD217, CD267, CD268, CD269, CD360, IL12RB2, IL23R, IL27RA, SIGIRR, TNFRSF25, CCRL2, CCR10, CD183, CD184, CD185, CD186, CD191, CD194, CD195, CD196, CD197, CXCR7, CX3CR1, and XCR1.

[0151] In some embodiments, the target protein is a cytokine receptor expressed on the cell surface of lymphocytes. For example, in some embodiments, the target protein may be selected from CD25, CD30, CD95, CD117, CD118, CD120a, CD120b, CD121a, CD121b, CD122, CD124, CD125, CD126, CD127, CD129, CD130, CD132, CD210A, CD210B, CD212, CD215, CD217, CD267, CD268, CD269, CD360, IL12RB2, IL23R, IL27RA, SIGIRR, and TNFRSF25.

[0152] In some embodiments, the target protein is a chemokine receptor expressed on the cell surface of lymphocytes. For example, in some embodiments, the target protein may be selected from CCRL2, CCR10, CD183, CD184, CD185, CD186, CD191, CD194, CD195, CD196, CD197, CXCR7, CX3CR1, and XCR1.

[0153] In some embodiments, the target protein is a subunit of the cytokine receptor interleukin-2 (IL-2) receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor, or IL-21 receptor. For example, in some embodiments, the target protein may be selected from CD25, CD122, CD124, CD127, CD129, CD132 (IL2RG), CD215, and CD360.

[0154] In some embodiments of the present invention, the target protein is interleukin-2 receptor subunit gamma (also known as IL2RG, cytokine receptor common subunit gamma, IL-2 receptor subunit gamma, IL-2R subunit gamma, IL-2RG, IL2Rγ, IL-2Rγ, and CD132). The IL2RG-blocking antibody REGN7257 effectively inhibits lymphocytes in vivo. See, e.g., WO 2020 / 160242 A1 and US2020 / 0247894 A1, which are hereby incorporated by reference in their entireties for all purposes. IL2RG is a subunit shared by several interleukin receptors, including IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R. The common cytokine receptor gamma chain was first identified as the third chain of the interleukin-2 (IL-2) receptor complex and named IL2RG. The same subunit was identified as part of several other cytokine receptor complexes: IL-4, IL-7, IL-9, IL-15, and IL-21, and thus can be referred to as γc (common cytokine receptor gamma chain). γc is involved in signal transduction and ligand binding of these cytokine receptors. Human IL2RG is assigned the UniProt accession number P31785. The canonical isoform of human IL2RG is assigned the UniProt accession number P31785-1 and the NCBI accession number NP_000197.1 and is set forth in SEQ ID NO:21. The engineered M145K variant of human IL2RG is set forth in SEQ ID NO:22. An exemplary messenger RNA encoding the canonical isoform of human IL2RG is assigned the NCBI accession number NM_000206.3 and is set forth in SEQ ID NO:23. The coding sequence of the canonical isoform of human IL2RG is assigned the CCDS ID CCDS14406.1 and is set forth in SEQ ID NO:24. The gene encoding human interleukin-2 receptor subunit gamma is called IL2RG, is located on the X chromosome, and is assigned the NCBI GeneID 3561. It is located at position Xq13.1 (reference genome version: GRCh38.p14 (GCF_000001405.40); position: NC_000023.11 (71107404..71111577, complementary strand).

[0155] The expression "functionally indistinguishable" means that the first and second subtypes can equally perform the same functions intracellularly without significant impairment. For example, the function can be binding to an endogenous ligand and / or activating a downstream signaling pathway intracellularly. In other words, the first and second subtypes are substantially functionally indistinguishable. In certain embodiments, minor functional impairment is acceptable. The substantially indistinguishable functions can be, for example, binding to an endogenous ligand and / or activating a downstream signaling pathway. In some embodiments, the function can be binding to an endogenous ligand and activating a downstream signaling pathway. The expression "immunologically distinguishable" means that the first and second subtypes of the protein can be distinguished by an antigen-binding protein that specifically binds to the first or second subtype but not the other, for example, an antigen-binding protein that specifically binds only to the second (unmodified) subtype of the target protein. In other words, the antigen-binding protein can distinguish the two subtypes by specifically binding only to one subtype and not the other. In a particular embodiment, an endogenous ligand binds (or binds to) the first and second subtypes (e.g., equally or only slightly impaired), but an engineered antigen-binding protein (e.g., an antibody) can distinguish the two subtypes by specifically binding only to one subtype and not the other (e.g., specifically binding only to the second subtype and not the first subtype).

[0156] In some embodiments, the second subtype of the target protein refers to the form present in a subject. In some embodiments, the second subtype of the target protein refers to the wild-type or natural form of the target protein (i.e., the form that typically occurs in nature), and the first subtype refers to the subtype obtained by introducing a mutation into the nucleic acid sequence encoding the second subtype. The natural form of a protein refers to the protein encoded by a nucleic acid sequence within the cell's genome and that has not been inserted or mutated by genetic manipulation (i.e., a natural protein is a protein that is not a non-transgenic protein nor a genetically engineered protein).

[0157] The mutations in the first subtype can be any type and any size of mutations. In some embodiments, the mutations include insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 1 - 20 amino acids, 1 - 5 amino acids, 1 - 3 amino acids, or 1 amino acid). In some embodiments, the mutations include substitutions (e.g., including substitution of 1 amino acid). In some embodiments, the mutations consist essentially of substitutions (e.g., consist essentially of substitution of 1 amino acid). In some embodiments, the mutations consist of substitutions (e.g., consist of substitution of 1 amino acid). The mutations can be at any site in the target protein. For example, if the target protein is a cell surface protein, then in some embodiments, the mutations can be in the extracellular domain of the target protein. In some embodiments, the mutation site can be a site that is not conserved among different mammalian species. In some embodiments, the mutations do not result in a change in the secondary structure of the surface protein. In some embodiments, the mutations are located within the epitope targeted by an antigen - binding protein or at a ligand - binding accessible site. In some embodiments, the mutations are not located at sites involved in protein - protein interactions of the surface protein that are predicted or experimentally established or confirmed to be involved in protein - protein interactions of the surface protein. In some embodiments, the mutations do not result in the deletion or introduction of intermolecular or intramolecular disulfide bond interactions or hydrophobic packing. In some embodiments, the mutations do not result in the deletion or introduction of post - translational protein modification sites, such as glycosylation sites. In some embodiments, based on crystal structure analysis or computer - aided structure prediction, the mutations are located at sites with a unique topology compared to other mammalian proteins. In the case where there is already an antibody or antigen - binding protein that reacts with the target protein, information about the epitope of the target protein recognized by the antibody or antigen - binding protein can be used to select the mutation site.

[0158] In some embodiments, the first subtype of the target protein is a genetically engineered subtype of the target protein. For example, the first subtype of the target protein can be genetically engineered to contain mutations (e.g., non-naturally occurring artificial mutations) to provide an altered epitope. For example, the altered epitope can be located in the binding region of an antigen-binding protein such as an antibody. In some embodiments, the target protein is IL2RG (e.g., human IL2RG), and the altered epitope is located in the binding region of the REGN7257 anti-IL2RG antibody described elsewhere herein. REGN7257 binding region 1 is encoded by exon 3 of IL2RG (e.g., human IL2RG) and includes T127, F128, V129, V130, Q131, L132, Q133, D134, P135, R136, E137, P138, R139, R140, Q141, A142, T143, Q144, M145, L146, K147, L148, Q149, and N150. REGN7257 binding region 2 is encoded by exons 2 and 3 of IL2RG (e.g., human IL2RG) and includes L87, H88, Y89 (exon 2), W90 (codon split between exons 2 and 3), Y91, K92, N93, S94, D95, N96, and D97 (exon 3). In some embodiments, the mutation can include a mutation (e.g., substitution) encoded by a nucleotide within exon 2 of the IL2RG gene (e.g., human IL2RG gene). In some embodiments, the mutation can include a mutation (e.g., substitution) encoded by a nucleotide within exon 3 of the IL2RG gene (e.g., human IL2RG gene). In some embodiments, the mutation can include a mutation (e.g., substitution) encoded by nucleotides within exons 2 and 3 of the IL2RG gene (e.g., human IL2RG gene). In some embodiments, the mutation can include a mutation (e.g., substitution) within the region from position T127 to position N150 of IL2RG (e.g., human IL2RG). In some embodiments, the mutation can include a mutation (e.g., substitution) within the region from position L87 to position D97 of IL2RG (e.g., human IL2RG). In some embodiments, the mutation can include a mutation (e.g., substitution) within the region from position T127 to position N150 of IL2RG (e.g., human IL2RG) and within the region from position L87 to position D97 of IL2RG (e.g., human IL2RG). In some embodiments, the mutation can include a mutation (e.g., substitution) within REGN7257 binding region 1 (SEQ ID NO:25). In some embodiments, the mutation can include a mutation (e.g., substitution) within REGN7257 binding region 2 (SEQ ID NO:62).In some embodiments, the mutations can include mutations (e.g., substitutions) within REGN7257 binding region 1 (SEQ ID NO:25) and within REGN7257 binding region 2 (SEQ ID NO:62). In some embodiments, the mutations can include mutations (e.g., substitutions) at one or more of the following positions: T127, F128, V129, V130, Q131, L132, Q133, D134, P135, R136, E137, P138, R139, R140, Q141, A142, T143, Q144, M145, L146, K147, L148, Q149, and N150. In some embodiments, the mutations can include mutations (e.g., substitutions) at one or more of the following positions: L87, H88, Y89, W90, Y91, K92, N93, S94, D95, N96, and D97. In some embodiments, the mutations can include mutations (e.g., substitutions) at one or more of the following positions: T127, F128, V129, V130, Q131, L132, Q133, D134, P135, R136, E137, P138, R139, R140, Q141, A142, T143, Q144, M145, L146, K147, L148, Q149, N150, L87, H88, Y89, W90, Y91, K92, N93, S94, D95, N96, and D97. For example, the mutations can include mutations (e.g., substitutions) at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, position K147, or any combination thereof within IL2RG (e.g., human IL2RG). A mutation at a position within IL2RG includes: a mutation (e.g., substitution) that includes only the residue at that position, or a mutation (e.g., substitution) that includes the residue at that position and other residues at other positions. The amino acid positions of the mutations or residues disclosed herein refer to the positions of the mutations or residues in the canonical isoform of human IL2RG shown in SEQ ID NO:21. In some embodiments, the mutation includes a mutation (e.g., substitution) at position M145. Examples of suitable M145 substitutions include M145K substitution, M145D substitution, M145E substitution, M145P substitution, M145W substitution, or M145Y substitution. In some embodiments, the mutation includes M145K substitution. In some embodiments, the mutation includes a mutation (e.g., substitution) at position W90. Examples of suitable W90 substitutions include W90V substitution, W90R substitution, W90Q substitution, W90L substitution, W90K substitution, W90E substitution, or W90D substitution. In some embodiments, the mutation includes W90Q substitution.In some embodiments, the mutations include mutations (e.g., substitutions) at positions M145 and W90.

[0159] The donor cells or edited cells can be administered to a subject by any suitable means. The term "administration" refers to the administration of a composition (e.g., donor cells or edited cells) to a subject or system (e.g., but not limited to, cells, organs, tissues, organisms, or a collection of their associated components or components). The route of administration may vary, e.g., depending on the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc. The term "administration" is intended to include the routes by which donor cells or edited cells are introduced into a subject to perform their intended function. In some embodiments, non-limiting examples of the routes of administration that can be used include, for example, injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), such as intravenous injection. In some embodiments of the present invention, the donor cells or edited cells are administered by intravenous injection. Administration may involve intermittent dosing or continuous dosing (e.g., but not limited to perfusion) over at least a selected period of time. The donor cells or edited cells can be administered alone, or in combination with another reagent (e.g., but not limited to, a reagent for selectively inhibiting or selectively depleting host cells or unedited cells in a subject), or in combination with a pharmaceutically acceptable carrier, or both. The donor cells or edited cells can be administered before, simultaneously with, or after the administration of other reagents.

[0160] Host cells or unedited cells in a subject can be selectively inhibited or selectively depleted based on their expression of a second subtype of the target protein by any suitable means. For example, they can be depleted or inhibited based on their expression of only the second subtype of the target protein and their lack of expression of the first subtype of the target protein. Alternatively, they can be depleted or inhibited based on their expression of the second subtype, regardless of the expression of the first subtype of their target protein. The selective inhibition or selective depletion of host cells or unedited cells can occur before, simultaneously with, or after the administration of the donor cells or edited cells. Selective depletion refers to the selective reduction of the total number or concentration of cells expressing a certain subtype of the target protein. The selective depletion of cells expressing the second subtype can correspond to the enrichment of cells expressing the first subtype.

[0161] In some embodiments, selective depletion refers to the selective ablation of host cells. Selective ablation of host cells refers to the ablation (i.e., killing) of host cells through an active killing mechanism. An active killing mechanism refers to a reagent directly killing host cells through a cytotoxic mechanism (e.g., antibody-drug conjugate (ADC), antibody-radioactive conjugate (ARC), CAR-T, or other engineered cytotoxicity), or recruiting host cell cytotoxic effector mechanisms (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP)), rather than blocking cell functions (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion; e.g., as in selective inhibition) without engaging exogenous cytotoxic effectors. In some embodiments, selective depletion of host cells or unedited cells includes the ablation of host cells or unedited cells through an active killing mechanism, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-drug conjugate (ADC), CAR-T, or other engineered cytotoxicity.

[0162] In other embodiments, selective depletion refers to the selective inhibition of host cells. In contrast to selective ablation, the selective inhibition of host cells does not include ablating (i.e., killing) host cells by an active killing mechanism. That is, the selective inhibition of host cells does not include cytotoxic ablation of host cells. In some embodiments, the selective inhibition of host cells or unedited cells does not include ablation or killing of host cells or unedited cells, even if carried out indirectly. In some embodiments, the selective inhibition or selective depletion of host cells or unedited cells includes: (1) blocking the growth of host cells or unedited cells (e.g., blocking proliferation or immune cell activation) to provide a competitive growth advantage for donor cells or edited cells; (2) blocking the localization or trafficking of host cells or unedited cells to provide a competitive homing advantage for donor cells or edited cells; (3) blocking the cell-cell interactions or adhesion of host cells or unedited cells to provide a competitive tissue infiltration advantage for donor cells or edited cells; or (4) blocking immune cell activation in host cells or unedited cells to provide a competitive advantage for donor cells or edited cells. For example, in some embodiments, the selective inhibition or selective depletion of host cells or unedited cells includes blocking growth (i.e., proliferation) and / or blocking immune cell activation. For example, in some embodiments, the selective inhibition or selective depletion of host cells or unedited cells includes blocking growth (i.e., proliferation) and blocking immune cell activation. For example, in some embodiments, the selective inhibition or selective depletion of host cells or unedited cells includes blocking growth (i.e., proliferation). For example, in some embodiments, the selective inhibition or selective depletion of host cells or unedited cells includes blocking immune cell activation. As a novel regulatory strategy, selectively inhibiting host cells without cytotoxic ablation has the potential to improve the safety and efficacy of cell therapy and transplantation therapy. Non-ablative regulation can avoid the adverse and harmful effects of ablating agents, including directly killing non-target cells expressing the drug target antigen (e.g., non-hematopoietic cells), indirect toxicity to adjacent tissues of the target, and long-term immunosuppression after transplantation. Selectively blocking or inhibiting essential host cell factors can provide a favorable competition for limiting host factors (e.g., cytokines, chemokines) and immune niche space, thereby enhancing the expansion, persistence, and trafficking of resistant donor cells without the use of stimulating and potentially toxic ablating agents. See, for example, Figures 2A - 5D .

[0163] In some embodiments of the present invention, the selective inhibition or selective depletion of host cells or unedited cells in a subject can include administering a reagent (e.g., an antagonist, an antigen-binding protein, or a population of cells expressing an antigen-binding protein (i.e., immune effector cells, such as chimeric antigen receptor T cells (CAR-T))) that specifically binds to a second subtype of a target protein but does not specifically bind to a first subtype of the target protein. For example, the reagent can be an antagonist that blocks the interaction between an endogenous ligand and the second subtype of the target protein but does not block the interaction between the endogenous ligand and the first subtype of the target protein. In some embodiments of the present invention, the selective inhibition or selective depletion of host cells or unedited cells in a subject can include administering an antagonist (e.g., an antigen-binding protein or a population of cells expressing an antigen-binding protein (i.e., immune effector cells, such as chimeric antigen receptor T cells (CAR-T))) that specifically binds to a second subtype of a target protein but does not specifically bind to a first subtype of the target protein. In some embodiments of the present invention, the selective inhibition or selective depletion of host cells or unedited cells in a subject can include administering an antigen-binding protein (e.g., an isolated antigen-binding protein) or one or more nucleic acids encoding an antigen-binding protein, such as an antibody (e.g., a human antibody, a monoclonal antibody, and / or a recombinant antibody) or an antigen-binding fragment thereof that specifically binds to a second subtype of a target protein (or an antigenic fragment thereof (e.g., an extracellular domain)) but does not specifically bind to the first subtype of the target protein. In some embodiments of the present invention, the selective inhibition or selective depletion of host cells or unedited cells in a subject can include administering a population of cells (i.e., immune effector cells) expressing an antigen-binding protein (e.g., a T cell expressing a chimeric antigen receptor or an exogenous T cell receptor), where the antigen-binding protein specifically binds to a second subtype of a target protein but does not specifically bind to the first subtype. An immune effector cell is a cell capable of effectuating or enhancing an immune response. In some embodiments (i.e., for selective ablation), selective depletion (e.g., selective ablation) can be achieved by a cytotoxic mechanism (e.g., an antibody-drug conjugate (ADC), an antibody-radioconjugate (ARC), CAR-T, or other engineered cytotoxicity). In some embodiments (i.e., for selective ablation), selective depletion (e.g., selective ablation) can be achieved by recruiting host cell cytotoxic effector mechanisms (e.g., complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP)), rather than by blocking cell function (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion; e.g., as in selective inhibition) without engaging an exogenous cytotoxic effector. In some embodiments (i.e., for selective ablation), the antigen-binding protein is conjugated to a toxin, thereby forming an immunotoxin. In some embodiments, the antigen-binding protein is not conjugated to a toxin.In some embodiments, the antigen-binding protein is a bispecific antigen-binding protein that can bind two different antigens simultaneously. In some embodiments, selective depletion (e.g., selective ablation) can be achieved by complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or antibody-drug conjugate (ADC). In some embodiments, selective inhibition or selective depletion is not achieved by a cytotoxic mechanism or by a mechanism that recruits host cell cytotoxic effectors. In some embodiments, selective inhibition or selective depletion is achieved by blocking cellular functions (e.g., growth or cytokine signaling, chemotactic tissue homing, cell-cell adhesion) without engaging exogenous cytotoxic effectors. In some embodiments, selective inhibition or selective depletion is not achieved by complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or antibody-drug conjugate (ADC). Toxins or drugs suitable for antibody-drug conjugates are well known in the art. See, e.g., Peters et al. (2015) Biosci. Rep. 35(4):e00225, Beck et al. (2017) Nature Reviews Drug Discovery 16:315-337; Marin-Acevedo et al. (2018) J. Hematol. Oncol. 11:8; Elgundi et al. (2017) Advanced Drug Delivery Reviews 122:2-19, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the antibody-drug conjugate can also comprise a linker (e.g., a peptide linker, e.g., a cleavable linker) that connects the antigen-binding protein (e.g., an antibody) and the drug molecule. Selective inhibition or selective depletion can also be achieved by administering an antigen-binding protein that is not conjugated to an effector compound (such as a drug or a toxin). In some embodiments, selective inhibition or selective depletion is achieved by blocking the binding of an endogenous ligand.

[0164] In methods of administering a reagent for selectively inhibiting or selectively depleting host cells or unedited cells, in some embodiments, the reagent can be administered concurrently with donor cells or edited cells. In some embodiments, the donor cells or edited cells are administered after the reagent. For example, in some embodiments, the donor cells or edited cells are administered within 1 day after the reagent, or at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or longer after the reagent. In some embodiments, the donor cells or edited cells are administered before the reagent. For example, in some embodiments, the donor cells or edited cells are administered within 1 day before the reagent, or at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or longer before the reagent.

[0165] In some embodiments, the donor cells or edited cells are administered multiple times (e.g., multiple doses). In some embodiments, the donor cells or edited cells are administered to a subject once. In some embodiments, the donor cells or edited cells are administered to a subject more than once (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more). In some embodiments, the donor cells or edited cells are administered to a subject at regular intervals (e.g., every 6 months). In methods of administering a reagent for selectively inhibiting or selectively depleting host cells or unedited cells, in some embodiments, the reagent can be administered in multiple administrations (e.g., multiple doses). In some embodiments, the reagent is administered to a subject once. In some embodiments, the reagent is administered to a subject more than once (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more). In some embodiments, the reagent is administered to a subject at regular intervals (e.g., every 6 months).

[0166] In some embodiments, a reagent is administered to a subject before and after administering a donor cell or an edited cell. In some embodiments, a reagent is administered to a subject before and after administering a donor cell or an edited cell, and the donor cell or the encoded cell is administered to the subject once. In some embodiments, for example, the reagent is administered to the subject about 1 to about 2 weeks before administering the donor cell or the edited cell and about 1 to 2 weeks after administering the donor cell or the edited cell (e.g., to give the donor or edited cell a competitive advantage).

[0167] In some embodiments, the reagent is administered to the subject multiple times (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more) before administering the donor cell or the edited cell. In some embodiments, the reagent is administered to the subject multiple times (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more) after administering the donor cell or the edited cell. In some embodiments, the reagent is administered to the subject multiple times (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more) before administering the donor cell or the edited cell and multiple times (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more) after administering the donor cell or the edited cell.

[0168] In some embodiments, about 10 6 to 10 11 donor cells or edited cells are administered. In some embodiments, it may be desirable to administer fewer than 10 6 cells to the subject. In some embodiments, it may be desirable to administer more than 10 11 cells to the subject. In some embodiments, one or more doses of cells comprise from about 10 6 cells to about 10 11 cells, from about 10 7 cells to about 10 10 cells, from about 10 8 cells to about 10 9 cells, from about 10 6 cells to about 10 8 cells, from about 10 7 cells to about 10 9 cells, from about 10 7 cells to about 10 10 cells, from about 10 7 cells to about 10 11 cells, from about 10 8 cells to about 10 10 cells, from about 108 from about 1 cell to about 10 11 cells, about 10 9 cells to about 10 10 cells, about 10 9 cells to about 10 11 cells, or about 10 10 cells to about 10 11 cells. In some embodiments, one or more doses of cells comprise about 10 6 to 10 7 cells / kg.

[0169] "Antagonist" includes a molecule that inhibits the activity of a target protein to any detectable extent. For example, an antagonist of IL2RG includes a molecule that inhibits IL2RG activity to any detectable extent (e.g., binding of a heteromeric receptor comprising IL2RG and a cytokine-specific receptor subunit complex to cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21).

[0170] In some embodiments, the reagent for selectively inhibiting or selectively depleting host cells or unedited cells is an antigen-binding protein.

[0171] The term "specifically binds or binds specifically" refers to an antigen-binding protein (such as an antibody or an antigen-binding fragment thereof) that has binding affinity for an antigen (such as the IL2RG protein), and the binding affinity is expressed as K D , at least about 10 -7 M (such as 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M), as measured by real-time label-free biolayer interferometry, for example at 25°C or 37°C, such as HTX biosensor, or by surface plasmon resonance, such as BIACORETM, or by solution affinity ELISA. In some embodiments, the antigen-binding protein used herein specifically binds to the IL2RG protein or the human IL2RG protein (e.g., wild-type or native IL2RG protein, e.g., wild-type or native human IL2RG protein). "Anti-IL2RG" refers to an antigen-binding protein (or another molecule) that specifically binds to IL2RG, such as an antibody or an antigen-binding fragment thereof.

[0172] An antigen is a molecule, such as a peptide (e.g., IL2RG or a fragment thereof (antigenic fragment)), to which an antibody binds, for example. The specific region on the antigen that an antibody recognizes and binds to is called an epitope.

[0173] The term "epitope" refers to an antigenic determinant (e.g., on IL2RG), which interacts with the specific antigen-binding site of an antigen-binding protein (e.g., the variable region of an antibody molecule, called a paratope). A single antigen may have multiple epitopes. Thus, different antigen-binding proteins (e.g., antibodies) can bind to different regions on the antigen and may have different biological effects. The term "epitope" can also refer to a site on an antigen (to which B and / or T cells respond), and / or an antigen region that binds to an antibody. Epitopes can be defined structurally or functionally. Functional epitopes are generally subsets of structural epitopes and have residues with an affinity that directly results in the interaction. Epitopes can be linear or conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope can include determinants that are the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural features and / or specific charge characteristics. The epitopes bound by the antigen-binding proteins used in the present invention can be contained in a fragment of IL2RG (e.g., human IL2RG), such as its extracellular domain, domain 1, or domain 2.

[0174] Methods for determining the epitopes of antigen-binding proteins (e.g., antibodies or fragments or polypeptides) include alanine scanning mutagenesis, peptide blotting (Reineke (2004) Methods Mol. Biol. 248:443-63, which is incorporated herein by reference in its entirety for all purposes), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be employed (Tomer (2000) Prot. Sci. 9:487-496, which is incorporated herein by reference in its entirety for all purposes). Another method for identifying amino acids within a polypeptide with which an antigen-binding protein (e.g., an antibody or fragment or polypeptide) interacts is hydrogen / deuterium exchange detected by mass spectrometry. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A, which are each incorporated herein by reference in their entirety for all purposes.

[0175] The term "antibody" as used herein refers to an immunoglobulin molecule that includes four polypeptide chains, two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds (i.e., a "complete antibody molecule") (e.g., IgG) - such as REGN7257 (also known as H4H12889P). In some embodiments, each antibody heavy chain (HC) includes a heavy chain variable region ("HCVR" or "V H”) (e.g., SEQ ID NO:2 or a variant thereof) and a heavy chain constant region (including domains C H 1, C H 2, and C H 3); and each antibody light chain (LC) includes a light chain variable region (“LCVR” or “V L ”) (e.g., SEQ ID NO:10 or a variant thereof) and a light chain constant region (C L ). The V H and V L regions can be further subdivided into hypervariable regions and more conserved regions that are interspersed, with the hypervariable regions being called complementarity-determining regions (CDRs) and the more conserved regions that are interspersed being called framework regions (FRs). Each V H and V L includes three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or antigen-binding fragment thereof) are identical to the germline sequences of a human, or are naturally or artificially modified.

[0176] Generally, the variable domains of immunoglobulin heavy and light chains each include three hypervariable regions, also called complementarity-determining regions (CDRs), which are located within relatively conserved framework regions (FRs). Generally, from the N-terminus to the C-terminus, the light and heavy chain variable domains each include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, amino acids are assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al., (1987) J. Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342:878-883, each of which is incorporated herein by reference in its entirety for all purposes. Thus, in some embodiments, the antigen-binding protein includes an antibody and antigen-binding fragments, including the CDRs of V H and the CDRs of V L , wherein V H and V LComprising the amino acid sequences (or variants thereof) described herein, wherein the CDRs are defined according to Kabat and / or Chothia.

[0177] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody or antigen-binding protein, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments (the heavy-chain portion of a Fab fragment cleaved with papain); (iv) Fv fragments (V H or V L ); and (v) single-chain Fv (scFv) molecules; composed of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, and small modular immunopharmaceuticals (SMIPs), are also included within the expression "antigen-binding fragment" as used herein. In some embodiments, the antigen-binding fragment comprises three or more CDRs of H4H12889P (e.g., CDR-H1, CDR-H2, and CDR-H3; or CDR-L1, CDR-L2, and CDR-L3).

[0178] In some embodiments, the antigen-binding protein is a "neutralizing" or "antagonistic" antigen-binding protein (e.g., an antibody or antigen-binding fragment) against a target protein, including a molecule that inhibits the activity of the target protein to any detectable extent (e.g., inhibits the binding of a receptor to one of its ligands).

[0179] In some embodiments, the antigen-binding protein may include monoclonal antigen-binding proteins, such as antibodies and their antigen-binding fragments, and monoclonal compositions comprising multiple isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" or "mAb" refers to members of a substantially homogeneous population of antibodies, i.e., the antibody molecules that make up the population are identical in amino acid sequence, except for possible minor naturally occurring mutations that may be present. The "multiple" such monoclonal antibodies and fragments in a composition refer to a concentration of the same (in terms of amino acid sequence, except for possible minor naturally occurring mutations) antibodies and fragments that is higher than that typically found in nature (e.g., in the blood of a host organism such as a mouse or a human).

[0180] In some embodiments, an antigen-binding protein, such as an antibody or an antigen-binding fragment, comprises a heavy-chain constant domain, such as IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4 (e.g., comprising S228P and / or S108P mutations)), or IgM types. In some embodiments, an antigen-binding protein, such as an antibody or an antigen-binding fragment, comprises a light-chain constant domain, such as κ or λ types. In some embodiments, the antigen-binding protein comprises an antigen-binding protein (e.g., H4H12889P) comprising the variable domains described herein, which variable domains are linked to the constant domains of the heavy and / or light chains, e.g., as described above.

[0181] In some embodiments, the antigen-binding protein is a human antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof, such as H4H12889P). As used herein, the term "human" antigen-binding protein, such as an antibody or an antigen-binding fragment, includes antibodies and fragments having variable and constant regions with immunoglobulin sequences derived from the human germline, whether in human cells or transplanted into non-human cells (e.g., mouse cells). See, e.g., US8502018, US6596541, or US5789215, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, human antibodies and antigen-binding fragments may include amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., having mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation), such as in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include such mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or non-human mammalian cells. The term is not intended to include antibodies isolated from or produced in a human subject.

[0182] In some embodiments, the antigen-binding protein is a chimeric antigen-binding protein (e.g., a chimeric antibody comprising the variable domains described herein (e.g., from H4H12889P)). As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, wherein the first and second antibodies are from different species. See, e.g., US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855, each of which is incorporated herein by reference in its entirety for all purposes.

[0183] In some embodiments, the antigen-binding protein is a recombinant antigen-binding protein (e.g., a recombinant antigen-binding protein as described herein, such as H4H12889P). The term "recombinant" antigen-binding protein, such as an antibody or an antigen-binding fragment thereof, refers to such molecules that are produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, such as transgenic mice), or host cells (such as Chinese hamster ovary (CHO) cells) or cell expression systems, or antibodies isolated from recombinant combinatorial human antibody libraries.

[0184] In some embodiments, the antigen-binding protein is an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of an antigen-binding protein as described herein, such as H4H12889P). In some embodiments, the antigen-binding fragment of the antibody will comprise at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one (e.g., 3) CDRs that are adjacent to or in-frame with one or more framework sequences. In an antigen-binding fragment having a V L domain associated with a V H domain, the V H and V L domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and comprise V H -V H 、V H -V L or V L -V L dimers. Alternatively, the antigen-binding fragment of the antibody can comprise non-covalently associated monomeric V H and / or V L domains.

[0185] In certain embodiments, the antigen-binding fragment of the antibody can comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragment of an antibody include: (i) V H -C H 1; (ii) V H -C H 2; OD V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of the variable and constant domains, including any of the exemplary configurations above, the variable and constant domains can be directly connected to each other or can be connected by a full or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, antigen-binding fragments of an antibody can contain homodimers or heterodimers (or other multimers) of any of the above variable and constant domain configurations, which bind non-covalently (e.g., via disulfide bonds) to each other and / or to one or more monomeric V H or V L domain structures.

[0186] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific), such as monospecific and multispecific (e.g., bispecific) antigen-binding fragments that contain one or more variable domains from antigen-binding proteins specifically described herein (e.g., H4H12889P).

[0187] In some embodiments, the antigen-binding protein is an antigen-binding protein that specifically binds to the IL2RG protein or an antigenic fragment thereof (e.g., the extracellular domain of IL2RG or human IL2RG), such as an antibody (e.g., a human antibody, a monoclonal antibody, or a recombinant antibody) or an antigen-binding fragment thereof. See, e.g., WO 2020 / 160242 A1 and US2020 / 0247894 A1, each of which is incorporated herein by reference in its entirety for all purposes. For example, the antigen-binding protein can comprise any polypeptide that includes the amino acid sequence set forth in SEQ ID NO: 18 and / or 20 or a variant thereof. Optionally, the antigen-binding protein comprises one or more other polypeptides, such as a human Fc (e.g., human IgG, such as IgG1 or IgG4 (e.g., comprising the S108P mutation)). Antigen-binding proteins that bind to the same epitope on IL2RG or that compete with any of the antigen-binding proteins described herein (e.g., H4H12889P or REGN7257) for binding to IL2RG can also be used.

[0188] In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) comprises an immunoglobulin heavy chain that comprises V H (e.g., HC) and / or an immunoglobulin light chain that comprises V L (e.g., LC), where V H comprises a combination of heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) shown respectively in SEQ ID NO: 4, 6, and 8, and V L comprises a combination of light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) shown respectively in SEQ ID NO: 12, 14, and 16.

[0189] In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) comprises an immunoglobulin heavy chain and light chain that respectively comprise V H (e.g., HC) and V L (e.g., LC), including a combination of heavy chain and light chain CDRs (CDR-H1, CDR-H2, and CDR-H3; and CDR-L1, CDR-L2, and CDR-L3) shown respectively in SEQ ID NO: 4, 6, 8, 12, 14, and 16.

[0190] In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) comprises a polypeptide pair that comprises the following V H and V L amino acid sequences: SEQ ID NO: 2 and 10.

[0191] In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) comprises the following pairs of amino acid sequences encoding the HC and LC: SEQ ID NO:18 and 20.

[0192] In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) comprises immunoglobulin V H and V L , or the HC and LC, which comprise variant amino acid sequences that have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, or 99%) total amino acid sequence identity or similarity to the corresponding V H , V L , HC, or LC amino acid sequences specifically described herein, but wherein the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of such immunoglobulins are not variants and comprise the amino acid sequences described herein. Thus, in these embodiments, the CDRs within the variant antigen-binding protein are not themselves variants.

[0193] In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) binds to the same epitope as H4H12889P. In some embodiments, the antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) competes with H4H12889P for binding to IL2RG. As used herein, the term "competes" refers to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) that binds to an antigen (e.g., IL2RG) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) to the antigen. Unless otherwise specified, the term also includes competition between two antigen-binding proteins (such as antibodies) in both directions, i.e., the first antibody binds to the antigen and blocks the second antibody from binding, and vice versa. Thus, in some embodiments, the competition occurs in one such direction. In certain embodiments, the first antigen-binding protein (e.g., an antibody) and the second antigen-binding protein (e.g., an antibody) can bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) can bind to different but e.g., overlapping or non-overlapping epitopes, wherein the binding of one antibody inhibits or blocks the binding of the second antibody, e.g., by steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, such as by real-time label-free biolayer interferometry. In addition, the binding competition between anti-IL2RG antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using real-time label-free biolayer interferometry on an Octet RED384 biosensor (Pall ForteBio Corp.).

[0194] In some embodiments, the antigen-binding protein is a variant of H4H12889P. Generally, an antibody or antigen-binding fragment modified in some way retains the ability to specifically bind to IL2RG. For example, when the activity is measured in moles, it retains at least 10% of the IL2RG-binding activity (compared to the parental antibody). Preferably, the antibody or antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the IL2RG-binding affinity of the parental antibody. It is also desirable that the antibody or antigen-binding fragment may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody), which do not substantially alter its biological activity.

[0195] A "variant" of such a polypeptide, such as an immunoglobulin chain (e.g., H4H12889P V H 、V L 、HC or LC or its CDR), refers to a polypeptide comprising an amino acid sequence that is at least about 70 - 99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) identical or similar to the reference amino acid sequence described herein (e.g., any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20); when compared by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the entire length of the corresponding reference sequence (e.g., expectation threshold: 10; word length: 3; maximum match in query range: 0; BLOSUM 62 matrix; gap cost: existence 11, extension 1; conditional combination score matrix adjustment).

[0196] In addition, variants of the polypeptide may include polypeptides, such as immunoglobulin chains (e.g., H4H12889P V H 、V L 、HC or LC or its CDR), which may include the amino acid sequence of the reference polypeptide specifically listed herein, but with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations, such as one or more missense mutations (e.g., conservative substitutions), nonsense mutations, deletions or insertions. For example, in some embodiments, an anti-IL2RG antigen-binding protein can be used, which includes an immunoglobulin light chain (or V L ) variant comprising the amino acid sequence described in SEQ ID NO: 10 but having one or more such mutations and / or an immunoglobulin heavy chain (or VH ) Variant. In some embodiments, the anti-IL2RG antigen-binding protein comprises an immunoglobulin light chain variant comprising CDR-L1, CDR-L2, and CDR-L3, wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more such mutations (e.g., conservative substitutions) and / or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2, and CDR-H3, wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more such mutations (e.g., conservative substitutions).

[0197] The following references relate to the BLAST algorithm, which is often used in sequence analysis: BLAST algorithm: Altschul et al. (2005) FEBS J. 272(20):5101-5109; Altschul et al. (1990) J. Mol. Biol. 215:403-410; Gish et al. (1993) Nature Genet. 3:266-272; Madden et al. (1996) Meth. Enzymol. 266:131-141; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang et al. (1997) Genome Res. 7:649-656; Wootton et al. (1993) Comput. Chem. 17:149-163; Hancock et al. (1994) Comput. Appl. Biosci. 10:67-70; Alignment scoring systems: Dayhoff et al. “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) Vol. 5, Suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R.M., et al., “Matrices for detecting distant relationships.” In Atlas of Protein Sequence and Structure, (1978) Vol. 5, Suppl. 3. "M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul (1991) J. Mol. Biol. 219:555-565; States et al. (1991) Methods 3:66-70; Henikoff et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919; Altschul et al. (1993) J. Mol. Evol. 36:290-300; Alignment statistics: Karlin et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268; Karlin et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:5873 - 5877; Dembo et al. (1994) Ann. Prob. 22:2022 - 2039; and Altschul, “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1 - 14, Plenum, N.Y., which are hereby incorporated by reference in their entirety for all purposes.

[0198] For example, a “conservative modification variant” or “conservative substitution” of an immunoglobulin chain as described herein refers to a variant in which one or more amino acids in a polypeptide are replaced by other amino acids having similar characteristics (such as charge, side - chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.). Such changes can often be made without significantly disrupting the biological activity of the antibody or fragment. Those skilled in the art recognize that, generally, a single amino acid substitution in a non - essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.), which is hereby incorporated by reference in its entirety for all purposes). In addition, substitutions of amino acids with similar structure or function are not likely to significantly disrupt biological activity. In some embodiments, an anti - IL2RG antigen - binding protein comprising such a conservative - modified variant immunoglobulin chain can be used.

[0199] Examples of groups of amino acids having side - chains with similar chemical properties include: (1) aliphatic side - chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side - chains: serine and threonine; (3) amide - containing side - chains: asparagine and glutamine; (4) aromatic side - chains: phenylalanine, tyrosine, and tryptophan; (5) basic side - chains: lysine, arginine, and histidine; (6) acidic side - chains: aspartic acid and glutamic acid, and (7) sulfur - containing side - chains: cysteine and methionine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log - likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 - 45, which is hereby incorporated by reference in its entirety for all purposes.

[0200] Unless otherwise indicated, "H4H12889P" (also known as REGN7257) refers to an anti-IL2RG antigen-binding protein (e.g., an antibody and its antigen-binding fragments (including multispecific antigen-binding proteins)) that respectively comprise an immunoglobulin heavy chain or its variable region (V H ), which comprises the amino acid sequence specifically described herein for H4H12889P (e.g., SEQ ID NO:2 (or a variant thereof)), and / or an immunoglobulin light chain or its variable region (V L ), which comprises the amino acid sequence specifically described herein for H4H12889P (e.g., SEQ ID NO:10 (or a variant thereof)); and / or comprises a heavy chain or V H , which comprises its CDRs (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof), and CDR-H3 (or a variant thereof)) and / or a light chain or V L , which comprises its CDRs (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof), and CDR-L3 (or a variant thereof)). In some embodiments, V H is linked to an IgG constant heavy chain domain, such as a human IgG constant heavy chain domain (e.g., IgG1 or IgG4 (e.g., comprising S228P and / or S108P mutations)) and / or V L is linked to a light chain constant domain, such as a human light chain constant domain (e.g., a λ or κ constant light chain domain). In some embodiments, polynucleotides encoding one or more of any such immunoglobulin chains (e.g., V H , V L , HC, and / or LC) are provided.

[0201] In some embodiments, the antigen-binding protein (e.g., an antibody and its antigen-binding fragments (e.g., H4H12889P)) comprises an immunoglobulin chain that includes the amino acid sequences specifically listed herein (and variants thereof) as well as cellular and in vitro post-translational modifications made to the antibody or fragment. For example, the antigen-binding protein includes an antibody and its antigen-binding fragments that specifically bind IL2RG and that comprise the heavy chain and / or light chain amino acid sequences described herein, and also includes antibodies and fragments in which one or more asparagine, serine, and / or threonine residues are glycosylated, one or more asparagine residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, the N-terminal glutamine is pyroglutamic acid (pyroE), and / or the C-terminal lysine or other amino acid is deleted.

[0202] In some embodiments, one or more nucleic acids encoding an antigen-binding protein are provided. The nucleic acid encoding the antigen-binding protein comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Such nucleic acids can be DNA, RNA, or a hybrid or derivative of DNA or RNA. Optionally, in some embodiments, the nucleic acid can be codon-optimized for efficient translation into a protein in a particular cell or organism. As a non-limiting example, compared to a naturally occurring polynucleotide sequence, the nucleic acid can be modified to substitute codons that are used more frequently in human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest. Any portion or fragment of the nucleic acid molecule can be generated by: (1) isolating the molecule from its natural environment; (2) using recombinant DNA techniques (such as but not limited to PCR amplification or cloning); or (3) using chemical synthesis methods. The nucleic acid can comprise modifications for enhancing stability or reducing immunogenicity. Non-limiting examples of modifications include: (1) alteration or replacement of one or two non-bridging phosphate oxygens and / or one or more bridging phosphate oxygens in the phosphodiester backbone linkage; (2) alteration or replacement of the components of the ribose sugar, such as alteration or replacement of the 2'-hydroxy group on the ribose sugar; (3) replacement of the phosphate moiety with a dephosphate linker; (4) modification or replacement of naturally occurring nucleobases; (5) replacement or modification of the ribose-phosphate backbone; (6) modification of the 3' or 5' end of the oligonucleotide (such as but not limited to removal, modification, or replacement of the terminal phosphate group or conjugation of a moiety); and (7) modification of the sugar.

[0203] Such nucleic acids can include any polynucleotide, such as an immunoglobulin V encoding H4H12889P H 、V L 、CDR-H, CDR-L, HC or LC polynucleotide; optionally, it is operably linked to a promoter or other expression control sequence. For example, such nucleic acids can include any polynucleotide (such as DNA) that comprises the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. In some embodiments, the polynucleotide of interest is fused to a secretion signal sequence.

[0204] In some embodiments, the nucleic acid can be in the form of an expression construct as defined elsewhere herein. As a non-limiting example, the nucleic acid can include regulatory regions (such as but not limited to transcriptional or translational control regions) that control the expression of the nucleic acid molecule, full-length or partial coding regions, and combinations thereof. As a non-limiting example, the nucleic acid can be operably linked to a promoter that is active in a cell or organism of interest. Promoters that can be used for such expression constructs include, for example, promoters that are active in one or more eukaryotic cells, such as mammalian cells (such as non-human mammalian cells or human cells), such as rodent cells (such as but not limited to mouse cells or rat cells). For example, these promoters can be conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters.

[0205] Generally, a "promoter" or "promoter sequence" is a DNA regulatory region that can bind RNA polymerase in a cell (e.g., directly or through other proteins or substances that bind to the promoter) and initiate transcription of a coding sequence. A promoter can be operably linked to other expression control sequences, including enhancer and repressor sequences and / or a polynucleotide encoding an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof). Promoters useful for controlling gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062, each incorporated herein by reference in its entirety for all purposes), the SV40 early promoter region (Benoist et al. (1981) Nature 290:304-310, incorporated herein by reference in its entirety for all purposes), the promoter contained in the Rous sarcoma virus 3' long terminal repeat (Yamamoto et al. (1980) Cell 22:787-797, incorporated herein by reference in its entirety for all purposes), the herpes thymidine kinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, incorporated herein by reference in its entirety for all purposes), the regulatory sequence of the metallothionein gene (Brinster et al. (1982) Nature 296:39-42, incorporated herein by reference in its entirety for all purposes); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731, incorporated herein by reference in its entirety for all purposes) or the tac promoter (DeBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25; see also "Useful Proteins from Recombinant Bacteria" in Scientific American (1980) 242:74-94, each incorporated herein by reference in its entirety for all purposes); and promoter elements from yeast or other fungi such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerate kinase) promoter, or the alkaline phosphatase promoter.

[0206] When, in a cell or other expression system, a polynucleotide encoding a polypeptide is "operably linked" to a promoter or other expression control sequence, that sequence directs RNA polymerase-mediated transcription of the coding sequence to RNA (preferably mRNA), which can then be spliced (if it contains introns) and optionally translated into the protein encoded by the coding sequence.

[0207] In some embodiments, one or more nucleic acids comprise the following encoding V Hand V L polynucleotide pairs: SEQ ID NO:1 and SEQ ID NO:9. In some embodiments, one or more nucleic acids comprise the following sets of polynucleotides encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3: SEQ ID NO:3, 5, 7, 11, 13, and 15. In some embodiments, one or more nucleic acids comprise the following polynucleotide pairs encoding HC and LC: SEQ ID NO:17 and 19. In some embodiments, one or more nucleic acids include a polynucleotide encoding an immunoglobulin polypeptide chain, which is a variant of those whose nucleotide sequences are specifically listed herein. A "variant" of such a polynucleotide or nucleic acid refers to a polynucleotide or nucleic acid that contains a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to the reference nucleotide sequence described herein; when compared by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the entire length of the corresponding reference sequence (e.g., expectation threshold: 10; word length: 28; maximum match within query range: 0; match / mismatch scores: 1, -2; gap cost: linear). In some embodiments, variants of the nucleotide sequences specifically listed herein contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) point mutations, insertions (e.g., in-frame insertions), or deletions (e.g., in-frame deletions) of one or more nucleotides. In some embodiments, such mutations may be missense or nonsense mutations. In some embodiments, such variant polynucleotides encode an immunoglobulin polypeptide chain that can be incorporated into an anti-IL2RG antigen-binding protein, i.e., such that the protein retains specific binding to IL2RG.

[0208] In some embodiments, the antigen-binding protein is an anti-IL2RG antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs, and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 12, 14, and 16, and wherein the three heavy chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 4, 6, and 8. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs, and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs are substantially respectively composed of sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 12, 14, and 16, and wherein the three heavy chain CDRs are substantially respectively composed of sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 4, 6, and 8. In some embodiments, the antigen-binding protein comprises an immunoglobulin light chain or its variable region containing three light chain CDRs, and an immunoglobulin heavy chain or its variable region containing three heavy chain CDRs, wherein the three light chain CDRs are respectively composed of sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 12, 14, and 16, and wherein the three heavy chain CDRs are respectively composed of sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 4, 6, and 8.

[0209] In some embodiments, the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, and the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8. In some embodiments, the three light chain CDRs are substantially respectively composed of the sequences shown in SEQ ID NO: 12, 14, and 16, and the three heavy chain CDRs are substantially respectively composed of the sequences shown in SEQ ID NO: 4, 6, and 8. In some embodiments, the three light chain CDRs are respectively composed of the sequences shown in SEQ ID NO: 12, 14, and 16, and the three heavy chain CDRs are respectively composed of the sequences shown in SEQ ID NO: 4, 6, and 8.

[0210] In some embodiments, the immunoglobulin light chain or its variable region comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, and the immunoglobulin heavy chain or its variable region comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:2. In some embodiments, the immunoglobulin light chain or its variable region consists essentially of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, and the immunoglobulin heavy chain or its variable region consists essentially of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:2. In some embodiments, the immunoglobulin light chain or its variable region consists of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, and the immunoglobulin heavy chain or its variable region consists of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:2.

[0211] In some embodiments, the immunoglobulin light chain or its variable region comprises the sequence shown in SEQ ID NO:10, and the immunoglobulin heavy chain or its variable region comprises the sequence shown in SEQ ID NO:2. In some embodiments, the immunoglobulin light chain or its variable region consists essentially of the sequence shown in SEQ ID NO:10, and the immunoglobulin heavy chain or its variable region consists essentially of the sequence shown in SEQ ID NO:2. In some embodiments, the immunoglobulin light chain or its variable region consists of the sequence shown in SEQ ID NO:10, and the immunoglobulin heavy chain or its variable region consists of the sequence shown in SEQ ID NO:2.

[0212] In some embodiments, the immunoglobulin light chain comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:20, and the immunoglobulin heavy chain comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:18. In some embodiments, the immunoglobulin light chain consists essentially of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:20, and the immunoglobulin heavy chain consists essentially of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:18. In some embodiments, the immunoglobulin light chain consists of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:20, and the immunoglobulin heavy chain consists of a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:18.

[0213] In some embodiments, the immunoglobulin light chain comprises the sequence shown in SEQ ID NO:20, and the immunoglobulin heavy chain comprises the sequence shown in SEQ ID NO:18. In some embodiments, the immunoglobulin light chain consists essentially of the sequence shown in SEQ ID NO:20, and the immunoglobulin heavy chain consists essentially of the sequence shown in SEQ ID NO:18. In some embodiments, the immunoglobulin light chain consists of the sequence shown in SEQ ID NO:20, and the immunoglobulin heavy chain consists of the sequence shown in SEQ ID NO:18.

[0214] Reagents for selectively inhibiting or selectively depleting host cells or unedited cells can be administered to a subject by any suitable means. The term administration refers to the administration of a composition to a subject or system (such as but not limited to cells, organs, tissues, organisms, or a collection of their associated components or components). The route of administration may vary, for example, depending on the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc. The term "administration" is intended to include the routes by which a reagent is introduced into a subject to perform its intended function. In some embodiments, non-limiting examples of the routes of administration that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), oral, inhalation, rectal, and transdermal. As non-limiting examples, administration to a subject (such as but not limited to humans or rodents) can be bronchial (including by bronchial instillation), oral, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and / or intravitreal. The reagent can be administered in the form of tablets or capsules (such as but not limited to by injection, inhalation, eye drops, ointments, suppositories, etc.), administered topically by a lotion or ointment, or administered rectally by a suppository. The administration can be a bolus or can be by continuous infusion. The administration may involve intermittent or continuous administration (such as but not limited to perfusion) over at least a selected period of time. Depending on the route of administration, the reagent can be coated or treated with a selected material to protect it from natural conditions that may adversely affect its ability to perform its intended function. The reagent can be administered alone, or in combination with another reagent (such as but not limited to the donor cells or edited cells described herein), or in combination with a pharmaceutically acceptable carrier, or in combination with both. The reagent can be administered before, simultaneously with, or after the administration of another reagent. In addition, the reagent can also be administered in a prodrug form that is converted in vivo to its active metabolite or a more active metabolite.

[0215] In some embodiments of the present invention, the subject can include, for example, any type of animal or mammal. Mammals include, for example, humans, non-human mammals, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (such as but not limited to mice, rats, hamsters, and guinea pigs), and livestock (such as but not limited to bovine species such as cows and steers; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human mammal" does not include humans. Specific non-limiting examples of non-human mammals include rodents, such as mice and rats. In some embodiments of the present invention, the subject is a human.

[0216] In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise generating donor cells or edited cells by modifying a cell population to express a first subtype of a target protein (e.g., the IL2RG protein). Suitable methods and reagents for generating donor cells or edited cells are described in more detail elsewhere herein. In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise isolating a cell population from the subject (or from a different subject) prior to modifying the cell population. In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise generating donor cells or edited cells by editing a target genomic locus (e.g., the IL2RG locus) in a cell population to express a first subtype of a target protein (e.g., the IL2RG protein). Suitable methods and reagents for editing the target genomic locus are described in more detail elsewhere herein. In some embodiments, the genomic locus is an endogenous genomic locus encoding the target protein (e.g., the target protein is IL2RG and the genomic locus is the IL2RG genomic locus). In some embodiments, the genomic locus is not an endogenous genomic locus encoding the target protein (e.g., the target protein is IL2RG and the genomic locus is not the IL2RG genomic locus). In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise isolating a cell population from the subject (or from a different subject) prior to editing the target genomic locus in the cell population. The isolated cells can be, for example, any suitable cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are lymphocytes or lymphoid progenitor cells. In some embodiments, the cells are T cells (e.g., CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells, γδ T cells, mucosal-associated invariant T cells (MAIT), tumor-infiltrating lymphocytes (TIL), or any combination thereof). In some embodiments, the cells are TIL. In some embodiments, the cells are B cells. In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are innate lymphoid cells. In some embodiments, the cells are dendritic cells. In some embodiments, the cells are hematopoietic stem cells (HSC), or hematopoietic stem and progenitor cells (HSPC), or their progeny.HSCs are capable of generating myeloid and lymphoid progenitors, which in turn give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphocytes (e.g., T cells, B cells, NK cells). In some embodiments, the cells are autologous (i.e., from the subject). In some embodiments, the cells are allogeneic (i.e., not from the subject) or syngeneic (i.e., genetically identical, or sufficiently identical and immunocompatible to permit transplantation). In some embodiments, the cells are mammalian cells or non-human mammalian cells (e.g., mouse or rat cells, or non-human primate cells) (e.g., the subject is a mammalian or non-human mammalian, and the cells are mammalian cells or non-human mammalian cells). In some embodiments, the cells are human cells (e.g., the subject is human, and the cells are human cells).

[0217] In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise, prior to administration to the subject, modifying a population of induced pluripotent stem cells (e.g., human induced pluripotent stem cells (iPSCs)) to express a first subtype of a target protein (e.g., the IL2RG protein), and then differentiating the induced pluripotent cells into different cell types, thereby generating donor cells or edited cells. In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise, prior to administration to the subject, editing a target genomic locus (e.g., the IL2RG locus) in a population of induced pluripotent stem cells (e.g., human induced pluripotent stem cells (iPSCs)) to express a first subtype of a target protein (e.g., the IL2RG protein), and then differentiating the induced pluripotent cells into different cell types, thereby generating donor cells or edited cells. For example, the induced pluripotent stem cells can differentiate into any suitable cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a lymphocyte or lymphoid progenitor. In some embodiments, the cell is a T cell (e.g., a CD4+ T cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a γδ T cell, a mucosa-associated invariant T cell (MAIT), a tumor-infiltrating lymphocyte (TIL), or any combination thereof). In some embodiments, the cell is a TIL. In some embodiments, the cell is a B cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is an innate lymphoid cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the cell is a hematopoietic stem cell (HSC), or a hematopoietic stem and progenitor cell (HSPC), or a descendant thereof. An HSC refers to a true stem cell that gives rise to all blood and immune lineages. HSPCs include HSCs but also more differentiated progenitors that give rise to more restricted lineages. For example, some HSPCs may only be able to develop into the myeloid lineage, or the lymphoid or erythroid lineages, etc. HSCs are capable of generating myeloid and lymphoid progenitors, which in turn give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, red blood cells, platelets, etc.) and lymphocytes (e.g., T cells, B cells, NK cells). In some embodiments, the donor cells or edited cells are autologous (i.e., from the subject). In some embodiments, the donor cells or edited cells are allogeneic (i.e., not from the subject) or syngeneic (i.e., genetically identical, or sufficiently identical and immunocompatible to permit transplantation).In some embodiments, the cell is a mammalian cell or a non-human mammalian cell (e.g., a mouse or rat cell, or a non-human primate cell) (e.g., the subject is a mammal or a non-human mammal, and the cell is a mammalian cell or a non-human mammalian cell). In some embodiments, the cell is a human cell (e.g., the subject is human, and the cell is a human cell).

[0218] In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise, prior to administration to the subject, modifying a population of hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs) (e.g., human HSCs or HSPCs) to express a first subtype of a target protein (e.g., the IL2RG protein), and then differentiating the HSCs or HSPCs into different cell types, thereby generating donor cells or edited cells. In some embodiments of the present invention, any method described herein for improving engraftment of donor cells or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further comprise, prior to administration to the subject, editing a target genomic locus (e.g., the IL2RG locus) in a population of hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs) (e.g., human HSCs or HSPCs) to express a first subtype of a target protein (e.g., the IL2RG protein), and then differentiating the HSCs or HSPCs into different cell types, thereby generating donor cells or edited cells. For example, the HSCs or HSPCs can differentiate into any suitable cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a lymphocyte or lymphoid progenitor cell. In some embodiments, the cell is a T cell (e.g., a CD4+ T cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a γδ T cell, a mucosa-associated invariant T cell (MAIT), a tumor-infiltrating lymphocyte (TIL), or any combination thereof). In some embodiments, the cell is a TIL. In some embodiments, the cell is a B cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is an innate lymphoid cell. In some embodiments, the cell is a dendritic cell. In some embodiments, the donor cells or edited cells are autologous (i.e., from the subject). In some embodiments, the donor cells or edited cells are allogeneic (i.e., not from the subject) or syngeneic (i.e., genetically identical, or sufficiently identical and immunocompatible to allow transplantation). In some embodiments, the cell is a mammalian cell or a non-human mammalian cell (e.g., a mouse or rat cell or a non-human primate cell) (e.g., the subject is a mammalian or non-human mammalian, and the cell is a mammalian cell or a non-human mammalian cell). In some embodiments, the cell is a human cell (e.g., the subject is human, and the cell is a human cell). III. Methods for Generating Donor Cells or Edited Cells

[0219] In some embodiments of the present invention, any method described herein for improving donor cell engraftment or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further include generating donor cells or edited cells. The donor cells or edited cells may be generated by modifying a cell population to express a first isoform of a target protein (e.g., the IL2RG protein). In some embodiments of the present invention, any method described herein for improving donor cell engraftment or for selectively inhibiting or selectively depleting host cells or unedited cells in a subject may further include generating donor cells or edited cells by editing a target genomic locus (e.g., the IL2RG locus) in a cell population to express a first isoform of a target protein (e.g., the IL2RG protein). The donor cells or edited cells may express only the first isoform, or they may express the first and second isoforms of the target protein (e.g., modified to express the first isoform of the target protein but retain expression of the second subunit of the target protein).

[0220] In some embodiments, generating a donor cell or an edited cell can include introducing an expression vector into a cell population, where the expression vector expresses a first isoform of a target protein (e.g., the IL2RG protein). Such an expression vector can contain the entire coding sequence of the first isoform of the target protein (e.g., IL2RG), operably linked to a promoter suitable for driving expression in the donor cell or the edited cell. Any suitable promoter can be used. In one instance, a promoter that is specific for or active in hematopoietic cells or subsets of hematopoietic cells can be used. In another instance, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken β-actin / CMV enhancer (CAG), and elongation factor-1α (EF1α). As another instance, an inducible promoter can be used. In some embodiments, the expression vector can be a bicistronic expression vector encoding a therapeutic molecule and the first isoform of the target protein (e.g., IL2RG) and a therapeutic molecule (e.g., a CAR), as described elsewhere herein. For examples of bicistronic constructs expressing a CAR and another molecule, see, e.g., Yeku et al. (2017) Sci. Rep. 7(1):10541 and Rafiq et al. (2018) Nat. Biotechnol. 36(9):847-856, which are hereby incorporated by reference in their entireties for all purposes. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector. In some embodiments, a lentiviral vector is used. In some embodiments, an AAV vector is used, such as an AAV vector having a serotype for expression in hematopoietic cells (e.g., AAV6). Optionally, the endogenous locus encoding the second isoform of the target protein can also be modified (e.g., disrupted) such that the first isoform is expressed but the second isoform is not expressed. As an example, the endogenous locus can be modified to contain an insertion, deletion, or one or more point mutations in the endogenous locus (e.g., the IL2RG locus), resulting in a loss of functional target protein (e.g., IL2RG) expression. Such a locus can contain a deletion or disruption of all endogenous coding sequences, or can contain a deletion or disruption of a fragment of the endogenous locus (i.e., a part of or a portion of). In one instance, the 5' fragment of the coding sequence can be deleted or disrupted (e.g., including the start codon). As an example, the endogenous locus can be modified such that the start codon of the endogenous locus is deleted or disrupted or mutated such that the start codon no longer functions. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substituting one or more nucleotides. In another instance, the 3' fragment of the endogenous locus can be deleted or disrupted (e.g., including the stop codon).In another instance, internal segments of the endogenous locus can be deleted or disrupted. In another instance, all coding sequences in the endogenous locus can be deleted or disrupted. Alternatively, the endogenous locus can remain unmodified and both the first and second subtypes are expressed.

[0221] In some embodiments, the donor cell or edited cell generated can include editing a genomic locus in a population of cells to express a first isoform of a target protein (e.g., the IL2RG protein). The genomic locus can be an endogenous locus encoding the target protein, it can be a safe harbor locus, or it can be a random genomic locus that is targeted by random integration. Safe harbor loci include chromosomal loci where transgenes or other exogenous nucleic acid inserts can be stably and reliably expressed in all tissues of interest without significantly altering cell behavior or phenotype (i.e., without any deleterious effects on the host cell). See, e.g., Sadelain et al. (2012) Nat. Rev. Cancer 12:51-58, which is incorporated herein by reference in its entirety for all purposes. For example, a safe harbor locus can be a locus where expression of the inserted gene sequence is not disrupted by any read-through expression from adjacent genes. For example, a safe harbor locus can include a chromosomal locus where exogenous DNA can be integrated and function in a predictable manner without adverse effects on endogenous gene structure or expression. Safe harbor loci can include extragenic or intragenic regions, e.g., loci within a gene that are non-essential, dispensable, or capable of being disrupted without obvious phenotypic consequences. Such safe harbor loci can provide an open chromatin configuration in all tissues and can be ubiquitously expressed during embryonic development and in the adult. See, e.g., Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:3789-3794, which is incorporated herein by reference in its entirety for all purposes. In addition, safe harbor loci can be efficiently targeted, and safe harbor loci can be disrupted without obvious phenotype. Examples of safe harbor loci include albumin, CCR5, HPRT, AAVS1, and Rosa26. See, e.g., U.S. Patent Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; and U.S. Patent Publications 2003 / 0232410; 2005 / 0208489; 2005 / 0026157; 2006 / 0063231; 2008 / 0159996; 2010 / 00218264; 2012 / 0017290; 2011 / 0265198; 2013 / 0137104; 2013 / 0122591; 2013 / 0177983; 2013 / 0177960; and 2013 / 0122591, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the genomic locus is an endogenous genomic locus encoding the target protein (e.g., the target protein is IL2RG and the genomic locus is the IL2RG genomic locus).In some embodiments, the genomic locus is not an endogenous genomic locus encoding the target protein (e.g., the target protein is IL2RG, and the genomic locus is not the IL2RG genomic locus). The coding sequence of the first subtype of the target protein (e.g., IL2RG) can be operably linked to a promoter suitable for driving expression in the donor cell or the edited cell. Optionally, the endogenous locus encoding the second subtype of the target protein can also be modified (e.g., disrupted) such that the first subtype is expressed, but the second subtype is not expressed. As an example, the endogenous locus can be modified to include an insertion, deletion, or one or more point mutations in the endogenous locus (e.g., the IL2RG locus), resulting in the loss of expression of the functional target protein (e.g., IL2RG). Such loci can include the deletion or disruption of all endogenous coding sequences, or can include the deletion or disruption of a fragment of the endogenous locus (i.e., a part of or a portion of). In one example, the 5' fragment of the coding sequence can be deleted or disrupted (e.g., including the start codon). As an example, the endogenous locus can be modified such that the start codon of the endogenous locus is deleted or disrupted or mutated, such that the start codon no longer functions. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substituting one or more nucleotides. In another example, the 3' fragment of the endogenous locus can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the endogenous locus can be deleted or disrupted. In another example, all coding sequences in the endogenous locus can be deleted or disrupted. Alternatively, the endogenous locus can remain unmodified and both the first and second subtypes are expressed.

[0222] In some embodiments, the resulting donor cell or edited cell can include the target genomic locus (e.g., the IL2RG locus) in the edited cell population to express the first subtype of the target protein (e.g., the IL2RG protein).

[0223] In some embodiments, editing can include introducing into a cell population: (1) a nuclease reagent or one or more nucleic acids encoding a nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in a target genomic locus, and (2) an exogenous donor nucleic acid. The nuclease can cleave the target genomic locus, and the exogenous donor nucleic acid can be inserted into the target genomic locus or can recombine with the target genomic locus to generate a donor cell or an edited cell that expresses a first subtype of the target protein. However, those skilled in the art know that alternative methods can also be used. For example, in some embodiments, base editors can be used to effect subtype conversion. See, e.g., Komor et al. (2016) Nature 533(7603):420-424, which is incorporated herein by reference in its entirety for all purposes. This method allows editing of the desired amino acids without the need to introduce double-strand DNA breaks.

[0224] Any suitable nuclease reagent can be used. In some embodiments, for example, these methods can utilize a nuclease reagent, such as a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system, a zinc finger nuclease (ZFN) system, or a transcription activator-like effector nuclease (TALEN) system, or components of such systems, to modify a target genomic locus (e.g., the IL2RG gene, e.g., the human IL2RG gene). Generally, a nuclease reagent involves the use of an engineered cleavage system to induce a double-strand break or a nick (i.e., a single-strand break) at a nuclease target site. The cleavage or nick can occur by using a specific nuclease (e.g., an engineered ZFN, TALEN, or CRISPR / Cas system) and an engineered guide RNA to direct specific cleavage or nicking of the nuclease target site. In the methods and compositions disclosed herein, any nuclease reagent that induces a nick or a double-strand break at a desired target sequence can be used. The nuclease reagent can be used to generate a targeted gene modification in the IL2RG gene (e.g., the human IL2RG gene). For example, in some embodiments, the targeted gene modification can include a targeted gene modification in exon 3 of IL2RG (e.g., exon 3 of human IL2RG). In some embodiments, the targeted gene modification is located in exon 3 of human IL2RG. For example, in some embodiments, the targeted gene modification can include a targeted gene modification in exon 2 of IL2RG (e.g., exon 2 of human IL2RG). In some embodiments, the targeted gene modification is located in exon 2 of human IL2RG. For example, in some embodiments, the targeted gene modification can include a targeted gene modification in exons 2 and 3 of IL2RG (e.g., exons 2 and 3 of human IL2RG). In some embodiments, the targeted gene modification is located in exons 2 and 3 of human IL2RG.

[0225] In some embodiments, the nuclease reagent is a CRISPR / Cas system. In some embodiments, the nuclease reagent comprises one or more ZFNs. In some embodiments, the nuclease reagent includes one or more TALENs.

[0226] The CRISPR / Cas system includes transcripts and other elements involved in Cas gene expression or directing Cas gene activity. The CRISPR / Cas system can be, for example, a type I, type II, type III system, or type V system (e.g., V-A subtype or V-B subtype). The methods and compositions disclosed herein can use the CRISPR / Cas system by utilizing a CRISPR complex (including a guide RNA (gRNA) complexed with a Cas protein) for site-specific binding or cleavage of nucleic acids. A CRISPR / Cas system targeting a target genomic locus includes a Cas protein (or a nucleic acid encoding a Cas protein) and one or more guide RNAs (or DNAs encoding one or more guide RNAs), wherein each of the one or more guide RNAs targets a different guide RNA target sequence in the target genomic locus.

[0227] The CRISPR / Cas systems used in the compositions and methods disclosed herein can be non-naturally occurring. Non-naturally occurring systems include anything that indicates human intervention, such as one or more components of the system being altered or mutated from their naturally occurring state, being at least substantially free of at least one other component with which they are naturally associated in nature, or being associated with at least one other component with which they are not naturally associated. For example, some CRISPR / Cas systems use non-naturally occurring CRISPR complexes that contain a gRNA and a Cas protein that do not naturally co-occur, or use a non-naturally occurring Cas protein, or use a non-naturally occurring gRNA.

[0228] The nuclease reagents and CRISPR / Cas systems described in the compositions and methods disclosed herein target a nuclease target sequence (e.g., a guide RNA target sequence) in a target genomic locus encoding a target protein. In some embodiments, the nuclease target sequence is located in the IL2RG gene. In some embodiments, the nuclease target sequence is located in the human IL2RG gene. In some embodiments, the nuclease target sequence is located in exon 3 of the IL2RG gene. In some embodiments, the nuclease target sequence is located in exon 3 of the human IL2RG gene. In some embodiments, the nuclease target sequence is located in exon 2 of the IL2RG gene. In some embodiments, the nuclease target sequence is located in exon 2 of the human IL2RG gene. In some embodiments, the nuclease target sequence is located in exons 2 and 3 of the IL2RG gene. In some embodiments, the nuclease target sequence is located in exons 2 and 3 of the human IL2RG gene.

[0229] Cas proteins. Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with a guide RNA. Cas proteins can also comprise a nuclease domain (e.g., a DNA nuclease domain or an RNA nuclease domain), a DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains. Some such domains (e.g., DNA nuclease domains) can be from native Cas proteins. Other such domains can be added to prepare modified Cas proteins. The nuclease domain has catalytic activity for nucleic acid cleavage, which includes the breaking of covalent bonds of nucleic acid molecules. The cleavage can produce blunt ends or staggered ends, and it can be single-stranded or double-stranded. For example, wild-type Cas9 protein generally produces blunt-end cleavage products. Alternatively, wild-type Cpf1 protein (e.g., FnCpf1) can produce cleavage products with a 5-nucleotide 5' overhang, where the cleavage occurs after the 18th base pair of the PAM sequence on the non-target strand and after the 23rd base on the target strand. Cas proteins can have full cleavage activity, producing a double-strand break (e.g., a double-strand break with blunt ends) at a target genomic locus, or it can be a nickase that produces a single-strand break at a target genomic locus.

[0230] Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or modified versions thereof.

[0231] Exemplary Cas proteins are Cas9 proteins or proteins derived from Cas9 proteins. Cas9 proteins come from type II CRISPR / Cas systems and generally share four key motifs with a conserved structure. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. Exemplary Cas9 proteins are from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.) Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.) Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Neisseria meningitidis, or Campylobacter jejuni. Other examples of Cas9 family members are described in WO 2014 / 131833, which is incorporated herein by reference in its entirety for all purposes. Cas9 from Streptococcus pyogenes (SpCas9) (e.g., assigned UniProt accession number Q99ZW2) is an exemplary Cas9 protein. An exemplary SpCas9 protein sequence is shown in SEQ ID NO: 41 (encoded by the DNA sequence shown in SEQ ID NO: 42). Smaller Cas9 proteins (e.g., Cas9 proteins such as: when combined with a guide RNA coding sequence and regulatory elements for Cas9 and the guide RNA, the coding sequence of the Cas9 protein is compatible with the maximum AAV packaging capacity, such as SaCas9 and CjCas9 and Nme2Cas9) are other exemplary Cas9 proteins. For example, Cas9 from Staphylococcus aureus (SaCas9) (e.g., assigned UniProt accession number J7RUA5) is another exemplary Cas9 protein. Similarly, Cas9 from Campylobacter jejuni (CjCas9) (e.g., assigned UniProt accession number Q0P897) is another exemplary Cas9 protein. See, e.g., Kim et al. (2017) Nat. Commun. 8:14500, which is incorporated herein by reference in its entirety for all purposes. SaCas9 is smaller than SpCas9, and CjCas9 is smaller than both SaCas9 and SpCas9. Cas9 from Neisseria meningitidis (Nme2Cas9) is another exemplary Cas9 protein. See, e.g., Edraki et al. (2019) Mol.Cell 73(4):714-726, which is incorporated herein by reference in its entirety for all purposes. Cas9 proteins from Streptococcus thermophilus (e.g., Streptococcus thermophilus LMD-9 Cas9 (St1Cas9) encoded by the CRISPR1 locus, or Streptococcus thermophilus Cas9 (St3Cas9) from the CRISPR3 locus) are other exemplary Cas9 proteins. Cas9 from Francisella novicida (FnCas9) or Francisella novicida Cas9 variants that recognize alternative PAMs (E1369R / E1449H / R1556A substitutions) are other exemplary Cas9 proteins. These and other exemplary Cas9 proteins are reviewed, for example, in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, which is incorporated herein by reference in its entirety for all purposes. Examples of Cas9 coding sequences, Cas9 mRNA, and Cas9 protein sequences are provided in WO 2013 / 176772, WO 2014 / 065596, WO 2016 / 106121, WO 2019 / 067910, WO 2020 / 082042, US 2020 / 0270617, WO 2020 / 082041, US2020 / 0268906, WO 2020 / 082046, and US 2020 / 0289628, each of which is incorporated herein by reference in its entirety for all purposes. Specific examples of ORF and Cas9 amino acid sequences are provided in Table 30 of paragraph

[0449] of WO 2019 / 067910, and specific examples of Cas9 mRNA and ORF are provided in paragraphs

[0214] -

[0234] of WO 2019 / 067910. See also Table 24 in WO 2020 / 082046 A2 (pages 84-85) and WO 2020 / 069296, each of which is incorporated herein by reference in its entirety for all purposes..

[0232] Another example of a Cas protein is Cpf1 (CRISPR from Prevotella and Francisella 1; Cas12a) protein. Cpf1 is a large protein (about 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9, as well as a counterpart to the characteristic arginine-rich cluster of Cas9. However, Cpf1 lacks the HNH nuclease domain present in the Cas9 protein, and the RuvC-like domain is continuous in the Cpf1 sequence, which is contrary to Cas9, which contains a long insertion including the HNH domain. See, e.g., Zetsche et al. (2015) Cell 163(3):759-771, which is incorporated herein by reference in its entirety for all purposes. Exemplary Cpf1 proteins are from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae.Cpf1 from Francisella novicida U112 (FnCpf1; assigned UniProt accession number A0Q7Q2) is an exemplary Cpf1 protein.

[0233] Another example of a Cas protein is CasX (Cas12e). CasX is an RNA-guided DNA endonuclease that produces staggered double-strand breaks in DNA. CasX is less than 1000 amino acids in size. Exemplary CasX proteins are from Deltaproteobacteria (DpbCasX or DpbCas12e) and Planctomycetes (PlmCasX or PlmCas12e). Like Cpf1, CasX uses a single RuvC active site for DNA cleavage. See, e.g., Liu et al. (2019) Nature 566(7743):218-223, which is incorporated herein by reference in its entirety for all purposes.

[0234] Another example of a Cas protein is CasΦ (CasPhi or Cas12j), which is uniquely present in phages. CasΦ is less than 1000 amino acids in size (e.g., 700-800 amino acids). CasΦ cleavage produces staggered 5' overhangs. The single RuvC active site in CasΦ is capable of crRNA processing and DNA cleavage. See, e.g., Pausch et al. (2020) Science 369(6501):333-337, which is incorporated herein by reference in its entirety for all purposes.

[0235] The Cas protein can be a wild-type protein (i.e., those that occur in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. In terms of the catalytic activity of a wild-type or modified Cas protein, the Cas protein can also be an active variant or fragment. In terms of catalytic activity, the active variant or fragment can have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the wild-type or modified Cas protein or a portion thereof, wherein the active variant retains the ability to cleave at the desired cleavage site, thereby retaining nick-inducing or double-strand break-inducing activity. Assay methods for nick-inducing or double-strand break-inducing activity are known and generally measure the overall activity and specificity of the Cas protein on a DNA substrate containing the cleavage site.

[0236] An example of a modified Cas protein is the modified SpCas9-HF1 protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9 with alterations (N497A / R661A / Q695A / Q926A) designed to reduce non-specific DNA contacts. See, e.g., Kleinstiver et al. (2016) Nature 529(7587):490-495, which is incorporated herein by reference in its entirety for all purposes. Another example of a modified Cas protein is the modified eSpCas9 variant (K848A / K1003A / R1060A), designed to reduce off-target effects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88, which is incorporated herein by reference in its entirety for all purposes. Other SpCas9 variants include K855A and K810A / K1003A / R1060A. These and other modified Cas proteins are reviewed, e.g., in Cebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, which is incorporated herein by reference in its entirety for all purposes. Another example of a modified Cas9 protein is xCas9, which is a SpCas9 variant that can recognize a broader range of PAM sequences. See, e.g., Hu et al. (2018) Nature 556:57-63, which is incorporated herein by reference in its entirety for all purposes.

[0237] Cas proteins can be modified to increase or decrease one or more of the following: nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. Cas proteins can also be modified to alter any other activity or property of the protein, such as stability. For example, one or more nuclease domains of a Cas protein can be modified, deleted, or inactivated, or the Cas protein can be truncated to remove domains that are not essential for protein function or to optimize (e.g., enhance or reduce) the activity or property of the Cas protein.

[0238] Cas proteins can comprise at least one nuclease domain, such as a DNase domain. For example, wild-type Cpf1 proteins typically comprise a RuvC-like domain that cuts both strands of the target DNA, possibly in a dimeric configuration. Similarly, CasX and CasΦ typically comprise a single RuvC-like domain that cuts both strands of the target DNA. Cas proteins can also comprise at least two nuclease domains, such as DNase domains. For example, wild-type Cas9 proteins typically comprise a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains can each cut a different strand of double-stranded DNA, thereby creating a double-strand break in the DNA. See, e.g., Jinek et al. (2012) Science 337(6096):816 - 821, which is incorporated herein by reference in its entirety for all purposes.

[0239] One or more nuclease domains can be deleted or mutated such that they no longer have function or have reduced nuclease activity. For example, if one nuclease domain in a Cas9 protein is deleted or mutated, the resulting Cas9 protein can be called a nickase and can create a single-strand break within double-stranded target DNA rather than a double-strand break (i.e., it can cut either the complementary or non-complementary strand, but not both). If no nuclease domain in the Cas9 protein is deleted or mutated, the Cas9 protein will retain double-strand break-inducing activity. An example of a mutation that converts Cas9 to a nickase is the D10A (aspartic acid at position 10 of Cas9 converted to alanine) mutation in the RuvC domain of Cas9 from Streptococcus pyogenes. Similarly, H939A (histidine at amino acid position 839 converted to alanine), H840A (histidine at amino acid position 840 converted to alanine), or N863A (asparagine at amino acid position N863 converted to alanine) in the HNH domain of Cas9 from Streptococcus pyogenes can convert Cas9 to a nickase. Other examples of mutations that convert Cas9 to a nickase include the corresponding mutations in Cas9 from Streptococcus thermophilus. See, e.g., Sapranauskas et al. (2011) Nucleic Acids Res. 39(21):9275 - 9282 and WO2013 / 141680, which are each incorporated herein by reference in their entirety for all purposes. Such mutations can be generated using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or total gene synthesis. Examples of other mutations that generate nickases can be found, e.g., in WO2013 / 176772 and WO 2013 / 142578, which are each incorporated herein by reference in their entirety for all purposes.

[0240] Examples of inactivating mutations in the catalytic domain of xCas9 are the same as those of SpCas9 above. Examples of inactivating mutations in the catalytic domain of Staphylococcus aureus Cas9 protein are also known. For example, the Staphylococcus aureus Cas9 enzyme (SaCas9) can contain a substitution at position N580 (e.g., N580A substitution) or a substitution at position D10 (e.g., D10A substitution) to produce a Cas nickase. See, e.g., WO2016 / 106236, which is incorporated herein by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domain of Nme2Cas9 are also known (e.g., D16A or H588A). Examples of inactivating mutations in the catalytic domain of St1Cas9 are also known (e.g., D9A, D598A, H599A or N622A). Examples of inactivating mutations in the catalytic domain of St3Cas9 are also known (e.g., D10A or N870A). Examples of inactivating mutations in the catalytic domain of CjCas9 are also known (e.g., D8A or H559A combination). Examples of inactivating mutations in the catalytic domain of FnCas9 and RHA FnCas9 are also known (e.g., N995A).

[0241] Examples of inactivating mutations in the catalytic domain of Cpf1 proteins are also known. For Cpf1 proteins from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella bovoculi 237 (MbCpf1Cpf1), such mutations can include mutations at position 908, 993, or 1263 of AsCpf1 or corresponding positions in Cpf1 orthologs, or mutations at position 832, 925, 947, or 1180 of LbCpfl or corresponding positions in Cpf1 orthologs. Such mutations can include, for example, one or more of the following: the mutations D908A, E993A, and D1263A of AsCpf1 or corresponding mutations in Cpf1 orthologs, or one or more of the mutations D832A, E925A, D947A, and D1180A of LbCpf1 or corresponding mutations in Cpf1 orthologs. See, for example, US2016 / 0208243, which is incorporated herein by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domain of CasX proteins are also known. For CasX proteins from Deltaproteobacteria, D672A, E769A, and D935A (alone or in combination) or corresponding positions in other CasX orthologs are inactivated. See, for example, Liu et al. (2019) Nature 566(7743):218 - 223, which is incorporated herein by reference in its entirety for all purposes. Examples of inactivating mutations in the catalytic domain of CasΦ proteins are also known. For example, D371A and D394A (alone or in combination) are inactivating mutations. See, for example, Pausch et al. (2020) Science 369(6501):333 - 337, which is incorporated herein by reference in its entirety for all purposes.

[0242] Cas proteins can also be operably linked to a heterologous polypeptide as a fusion protein. For example, a Cas protein can be fused to a cleavage domain. See WO 2014 / 089290, which is incorporated herein by reference in its entirety for all purposes. A Cas protein can also be fused to a heterologous polypeptide to provide increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internally of the Cas protein.

[0243] As an example, a Cas protein can be fused to one or more heterologous polypeptides that provide subcellular localization. These heterologous polypeptides can include, for example, one or more nuclear localization signals (NLSs), such as the monopartite SV40 NLS and / or bipartite α-importin NLS for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, an ER retention signal, etc. See, e.g., Lange et al. (2007) J. Biol. Chem. 282(8):5101 - 5105, which is incorporated herein by reference in its entirety for all purposes. These subcellular localization signals can be located at the N-terminus, C-terminus, or anywhere internal to the Cas protein. An NLS can comprise a stretch of basic amino acids and can be a monopartite sequence or a bipartite sequence. Optionally, a Cas protein can comprise two or more NLSs, including an N-terminal NLS (e.g., an α-importin NLS or monopartite NLS) and a C-terminal NLS (e.g., an SV40 NLS or bipartite NLS). A Cas protein can also comprise two or more NLSs at the N-terminus and / or two or more NLSs at the C-terminus.

[0244] For example, a Cas protein can be fused to 1 - 10 NLSs (e.g., fused to 1 - 5 NLSs or to one NLS). When using one NLS, the NLS can be linked at the N-terminus or C-terminus of the Cas protein sequence. It can also be inserted internal to the Cas protein sequence. Alternatively, a Cas protein can be fused to multiple NLSs. For example, a Cas protein can be fused to 2, 3, 4, or 5 NLSs. In a particular example, a Cas protein can be fused to two NLSs. In some cases, the two NLSs can be the same (e.g., two SV40 NLSs) or different. For example, a Cas protein can be fused to two SV40 NLS sequences linked at the carboxyl terminus. Alternatively, a Cas protein can be fused to two NLSs, one linked at the N-terminus and one at the C-terminus. In other examples, a Cas protein can be fused to 3 NLSs or not fused to an NLS. An NLS can be a monopartite sequence, such as the SV40 NLS, PKKKRKV (SEQ ID NO:43), or PKKKRRV (SEQ ID NO:44). An NLS can be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO:45). In a particular example, a single PKKKRKV (SEQ ID NO:43) NLS can be linked at the C-terminus of the Cas protein. One or more linkers are optionally included at the fusion site.

[0245] The Cas protein can also be operably linked to a cell-penetrating domain or a protein transduction domain. For example, the cell-penetrating domain can be derived from the HIV-1 TAT protein, the TLM cell-penetrating motif from the human hepatitis B virus, MPG, Pep-1, VP22, the cell-penetrating peptide from herpes simplex virus, or the polyarginine peptide sequence. See, e.g., WO 2014 / 089290 and WO 2013 / 176772, which are hereby incorporated by reference in their entirety for all purposes. The cell-penetrating domain can be located at the N-terminus, C-terminus, or anywhere internally of the Cas protein.

[0246] The Cas protein can be provided in any form. For example, the Cas protein can be provided in protein form, such as the Cas protein complexed with gRNA. Alternatively, the Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Cas protein can be codon-optimized for efficient translation into protein in a particular cell or organism. For example, compared to the naturally occurring polynucleotide sequence, the nucleic acid encoding the Cas protein can be modified to replace codons with those that are used more frequently in bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest. When the nucleic acid encoding the Cas protein is introduced into a cell, the Cas protein can be transiently, conditionally, or constitutively expressed in the cell.

[0247] The nucleic acid encoding the Cas protein can be stably integrated into the genome of the cell and operably linked to a promoter that is active in the cell. Alternatively, the nucleic acid encoding the Cas protein can be operably linked to a promoter in an expression construct. An expression construct includes any nucleic acid construct capable of directing the expression of a gene or other nucleic acid sequence of interest (such as the Cas gene) and capable of transferring such nucleotide sequence of interest to a target cell. For example, the nucleic acid encoding the Cas protein can be in a vector that contains DNA encoding the gRNA. Alternatively, it can be in a vector or plasmid that is separate from the vector containing DNA encoding the gRNA. Promoters that can be used for the expression construct include, for example, promoters that are active in human cells or human hematopoietic cells. For example, these promoters can be conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Optionally, the promoter can be a bidirectional promoter that drives the expression of the Cas protein in one direction and the expression of the guide RNA in the other direction. Such a bidirectional promoter can consist of: (1) a complete, conventional, unidirectional PolIII promoter that contains three external control elements: a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box; and (2) a second basal Pol III promoter that includes the PSE and a TATA box fused to the 5' end of the DSE in the opposite direction. For example, in the H1 promoter, the DSE is adjacent to the PSE and the TATA box, and by creating a hybrid promoter, the promoter can be made bidirectional, where the reverse transcription is controlled by appending the PSE and TATA box derived from the U6 promoter. See, for example, US 2016 / 0074535, which is incorporated herein by reference in its entirety for all purposes. Using a bidirectional promoter to simultaneously express the genes encoding the Cas protein and the guide RNA can generate a compact expression cassette to facilitate delivery. In certain embodiments, the promoter is acceptable to regulatory agencies for use in humans. In certain embodiments, the promoter drives expression in hematopoietic cells.

[0248] Different promoters can be used to drive Cas expression or Cas9 expression. In some methods, small promoters are used so that the Cas or Cas9 coding sequence can fit into the AAV construct. For example, Cas or Cas9 and one or more gRNAs (e.g., 1 gRNA or 2 gRNAs or 3 gRNAs or 4 gRNAs) can be delivered by LNP-mediated delivery (e.g., in the form of RNA) or adeno-associated virus (AAV)-mediated delivery (e.g., AAV8-mediated delivery). For example, the nuclease reagent can be CRISPR / Cas9, and Cas9 mRNA and gRNA (e.g., targeting the IL2RG gene (e.g., the human IL2RG gene)) can be delivered by LNP-mediated delivery or AAV-mediated delivery. Cas or Cas9 and one or more gRNAs can be delivered in a single AAV or by two separate AAVs. For example, the first AAV can carry the Cas or Cas9 expression cassette, and the second AAV can carry the gRNA express...

Claims

1. A method for improving engraftment of donor cells in a subject in need thereof, comprising: (a) providing donor cells that have been modified to express a first subtype of a target protein, wherein the target protein is a protein expressed on the cell surface of hematopoietic cells, wherein the first subtype of the target protein is different from a second subtype of the target protein, and wherein the second subtype is expressed in host cells of the subject; (b) administering the donor cells to the subject, and (c) selectively inhibiting host cells in the subject based on the expression of the second subtype of the target protein by the host cells, thereby improving engraftment of the donor cells in the subject.

2. The method according to claim 1, wherein the target protein is a receptor.

3. The method according to claim 1 or 2, wherein the target protein is a cytokine receptor or a chemokine receptor, optionally, wherein the target protein is the cytokine receptor.

4. The method according to any one of the preceding claims, wherein the target protein is a protein expressed on the cell surface of lymphocytes.

5. The method according to any one of the preceding claims, wherein the target protein is a subunit of a cytokine receptor interleukin-2 (IL-2) receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-15 receptor or IL-21 receptor.

6. The method according to any one of the preceding claims, wherein the target protein is interleukin-2 receptor subunit gamma (IL2RG).

7. The method according to any one of the preceding claims, wherein the selective inhibition of the host cells in step (c) does not include ablating the host cells by an active killing mechanism.

8. The method according to any one of the preceding claims, wherein the selective inhibition in step (c) comprises: (1) Blocking the growth of the host cells to provide a competitive growth advantage for the donor cells; (2) Blocking the localization or transport of the host cells to provide a competitive homing advantage for the donor cells; (3) Blocking the cell-cell interaction or adhesion of the host cells to provide a competitive tissue infiltration advantage for the donor cells; or (4) Blocking the activation of immune cells in the host cells to provide a competitive advantage for the donor cells.

9. The method according to any one of the preceding claims, wherein the selective inhibition in step (c) includes blocking the growth of the host cells and / or blocking the activation of immune cells in the host cells to provide a competitive growth advantage for the donor cells.

10. The method according to any one of the preceding claims, wherein the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable.

11. The method according to any one of the preceding claims, wherein the donor cells express the first subtype of the target protein and the second subtype of the target protein.

12. The method according to any one of claims 1-10, wherein the donor cells express only the first subtype of the target protein.

13. The method according to any one of the preceding claims, wherein the first subtype of the target protein is expressed from an expression vector in the donor cells, or wherein the genomic locus has been edited to express the first subtype of the target protein in the donor cells.

14. The method according to claim 13, wherein the genomic locus is an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is the IL2RG genomic locus.

15. The method according to claim 13, wherein the genomic locus is not an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is not the IL2RG genomic locus.

16. The method according to any one of the preceding claims, wherein the first subtype of the target protein is a genetically engineered subtype of the target protein.

17. The method according to any one of the preceding claims, wherein the first subtype of the target protein is genetically engineered to contain a mutation, thereby providing an altered epitope, optionally, wherein the mutation is an artificial mutation.

18. The method according to claim 17, wherein the altered epitope is a binding region of a target protein antagonist, such that the target protein antagonist exhibits a reduced or eliminated ability to bind and / or inhibit the first subtype of the target protein as compared to its ability to bind and / or inhibit the second subtype of the target protein.

19. The method according to claim 18, wherein both the first subtype of the target protein and the second subtype of the target protein retain the ability to bind an endogenous ligand, optionally, wherein the target protein antagonist blocks the binding of the endogenous ligand to the second subtype of the target protein, but does not block the binding of the endogenous ligand to the first subtype of the target protein.

20. The method according to claim 18 or 19, wherein the target protein is IL2RG, wherein the altered epitope is in the binding region of the target protein antagonist, wherein the target protein antagonist is an antibody, which comprises an immunoglobulin light chain or its variable region, which comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14 and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6 and 8, consist essentially of or consist of the same.

21. The method according to any one of claims 17-20, wherein the target protein is IL2RG, and the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus.

22. The method according to any one of claims 17-21, wherein the target protein is IL2RG, and the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97.

23. The method according to any one of claims 17 - 22, wherein the target protein is IL2RG, and the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147.

24. The method according to any one of claims 17 - 23, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position M145, optionally, wherein the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution.

25. The method according to any one of claims 17 - 24, wherein the target protein is IL2RG, and wherein the mutation comprises an M145K substitution.

26. The method according to any one of claims 17 - 25, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position W90, optionally, wherein the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution, optionally, wherein the mutation comprises a W90Q substitution.

27. The method according to any one of claims 17 - 26, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

28. The method according to any of the preceding claims, wherein the selective inhibition in step (c) comprises administering a target protein antagonist to the subject, wherein the target protein antagonist specifically binds to the second subtype of the target protein but does not specifically bind to the first subtype of the target protein, optionally, wherein step (c) comprises multiple administrations of the target protein antagonist.

29. The method according to any one of claims 18 - 20 and 28, wherein the target protein antagonist is an antigen - binding protein.

30. The method according to claim 29, wherein the antigen - binding protein is an antibody or an antigen - binding fragment thereof.

31. The method according to claim 29 or 30, wherein the target protein is IL2RG, wherein the antigen - binding protein comprises an immunoglobulin light chain or its variable region, which comprises three light - chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy - chain CDRs, wherein the three light - chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NO:12, 14, and 16, consists essentially of or consists of the same, and wherein the three heavy - chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NO:4, 6, and 8, consists essentially of or consists of the same.

32. The method according to claim 31, wherein the three light chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NO: 12, 14, and 16, and wherein the three heavy chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NO: 4, 6, and 8.

33. The method according to claim 29 or 30, wherein the target protein is IL2RG, wherein the antigen-binding protein comprises an immunoglobulin light chain variable region that comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of, or consists of the sequence, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain variable region that comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of, or consists of the sequence.

34. The method according to claim 33, wherein the target protein is IL2RG, wherein the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consists essentially of, or consists of the sequence, and wherein the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consists essentially of, or consists of the sequence.

35. The method according to claim 29 or 30, wherein the target protein is IL2RG, wherein the antigen-binding protein comprises an immunoglobulin light chain that comprises the sequence shown in SEQ ID NO: 20, consists essentially of, or consists of the sequence, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain that comprises the sequence shown in SEQ ID NO: 18, consists essentially of, or consists of the sequence.

36. The method according to any one of the preceding claims, wherein the donor cell and / or the host cell is a hematopoietic cell, optionally wherein the donor cell and / or the host cell is an immune cell.

37. The method according to any one of the preceding claims, wherein the donor cell and / or the host cell is a lymphocyte or lymphoid progenitor cell.

38. The method according to any one of the preceding claims, wherein the donor cell and / or the host cell is a T cell.

39. The method according to any one of the preceding claims, wherein the donor cell and / or the host cell is a tumor-infiltrating lymphocyte (TIL).

40. The method according to any one of claims 1-37, wherein the donor cell and / or the host cell is a B cell.

41. The method according to any one of claims 1-37, wherein the donor cell and / or the host cell is a NK cell.

42. The method according to any one of claims 1-36, wherein the donor cell and / or the host cell is a hematopoietic stem and progenitor cell.

43. The method according to any one of claims 1-41, wherein the donor cell and / or the host cell is derived from a hematopoietic stem cell, or a hematopoietic stem and progenitor cell.

44. The method according to any one of claims 1-42, wherein the donor cell is derived from induced pluripotent stem cells.

45. The method according to any one of the preceding claims, wherein the subject is a mammal or a non-human mammal, and the donor cell is a mammalian cell or a non-human mammalian cell.

46. The method according to any one of the preceding claims, wherein the subject is a human, and the donor cell is a human cell.

47. The method according to any one of the preceding claims, wherein the donor cell comprises or expresses a therapeutic molecule.

48. The method according to claim 47, wherein the therapeutic molecule does not target the target protein.

49. The method according to any one of the preceding claims, wherein the donor cell comprises or expresses an immunoglobulin.

50. The method according to claim 49, wherein the immunoglobulin does not target the target protein.

51. The method according to any one of the preceding claims, wherein the donor cell comprises a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

52. The method according to claim 51, wherein the CAR or the exogenous TCR does not target the target protein.

53. The method according to any one of the preceding claims, wherein the donor cell is autologous.

54. The method according to any one of claims 1-52, wherein the donor cell is allogeneic or syngeneic.

55. The method according to any one of the preceding claims, wherein the subject has a disease or disorder, and the method is used to treat the disease or disorder.

56. The method according to any one of the preceding claims, wherein the subject has cancer.

57. The method according to claim 56, wherein the cancer is a solid tumor cancer.

58. The method according to claim 56, wherein the cancer is a hematological cancer.

59. The method according to any one of the preceding claims, wherein the subject has a hematopoietic malignancy, and the method is used to treat the subject's hematopoietic malignancy.

60. The method according to any one of the preceding claims, wherein the subject has defective immune cells or a hematopoietic genetic defect.

61. The method according to claim 60, wherein the hematopoietic genetic defect is sickle cell disease or severe combined immunodeficiency (SCID).

62. The method according to any one of the preceding claims, wherein steps (b) and (c) occur simultaneously.

63. The method according to any one of claims 1-61, wherein step (b) occurs before step (c), optionally, wherein step (c) comprises multiple administrations of a target protein antagonist after step (b).

64. The method according to any one of claims 1-61, wherein step (b) occurs after step (c), optionally, wherein step (c) comprises multiple administrations of a target protein antagonist before step (b).

65. The method according to any one of claims 1-64, wherein step (c) occurs before and after step (b), optionally, wherein step (c) comprises multiple administrations of a target protein antagonist before step (b) and / or multiple administrations of a target protein antagonist after step (b).

66. The method according to any one of the preceding claims, further comprising generating the donor cell by modifying a cell population to express a first subtype of the target protein prior to step (a).

67. The method according to claim 66, wherein the cell population is a population of induced pluripotent stem cells, and the method further comprises differentiating the induced pluripotent stem cells into the donor cell administered in step (a) prior to step (a), optionally, wherein the induced pluripotent stem cells are differentiated into hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells, NK cells, hematopoietic stem cells, or hematopoietic stem cells and progenitor cells.

68. The method according to claim 66, wherein the cell population is a population of hematopoietic stem cells, or a population of hematopoietic stem cells and progenitor cells, and the method further comprises differentiating the hematopoietic stem cells or hematopoietic stem cells and progenitor cells into the donor cell administered in step (a) prior to step (a), optionally, wherein the hematopoietic stem cells or hematopoietic stem cells and progenitor cells are differentiated into differentiated hematopoietic cells, lymphocytes or lymphoid progenitor cells, T cells, B cells or NK cells.

69. The method according to any one of claims 66-68, wherein generating the donor cell comprises introducing an expression vector encoding the first subtype of the target protein to express the first subtype of the target protein prior to step (a), or wherein generating the donor cell comprises editing a genomic locus in the cell population to express the first subtype of the target protein prior to step (a).

70. The method according to claim 69, wherein the genomic locus is an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is the IL2RG genomic locus.

71. The method according to claim 69, wherein the genomic locus is not an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is not the IL2RG genomic locus.

72. The method according to any one of claims 69-71, wherein the editing comprises introducing into the cell population: (1) a nuclease reagent or one or more nucleic acids encoding the nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in the genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the genomic locus, and the exogenous donor nucleic acid inserts into or recombines with the genomic locus to generate the donor cell expressing the first subtype of the target protein.

73. The method according to claim 72, wherein the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c)(i) a Cas protein; and (ii) A guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that is a nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

74. The method according to claim 73, wherein the nuclease reagent comprises the Cas protein and the guide RNA. Optionally, wherein the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 76-87, or the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 64-75, or optionally, wherein the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 136-153, or the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 118-135.

75. The method according to claim 73 or 74, wherein the Cas protein is a Cas9 protein.

76. The method according to any one of claims 72-75, wherein the exogenous donor nucleic acid comprises homology arms.

77. The method according to any one of claims 72-76, wherein the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN). Optionally, wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 88-117, or optionally, wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 154-173.

78. The method according to claim 77, wherein: (I) The DNA targeting fragment comprises the sequence shown in SEQ ID NO: 77, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO: 65, and wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 88-97; (II) The DNA targeting fragment comprises the sequence shown in SEQ ID NO: 83, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO: 71, and wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 98-107; (III) The DNA targeting fragment comprises the sequence shown in SEQ ID NO: 86, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO: 74, and wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 108-117; (IV) The DNA targeting fragment comprises the sequence shown in SEQ ID NO: 137, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO: 119, and wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 154-163; or (V) The DNA targeting fragment comprises the sequence shown in SEQ ID NO: 138, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO: 120, and wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 164 - 173.

79. The method according to any one of claims 66 - 78, further comprising isolating the cell population from the subject or a different subject prior to modifying the cell population.

80. A combination medicament for administration to a subject in need thereof, comprising: (a) A population of donor cells modified to express a first subtype of a target protein, wherein the target protein is a protein expressed on the cell surface of hematopoietic cells, and wherein the first subtype of the target protein is different from the second subtype of the target protein; and (b) A target protein antagonist that specifically binds to the second subtype of the target protein but does not specifically bind to the first subtype of the target protein.

81. The combination medicament according to claim 80, wherein the target protein is a receptor.

82. The combination medicament according to claim 80 or 81, wherein the target protein is a cytokine receptor or a chemokine receptor, optionally, wherein the target protein is the cytokine receptor.

83. The combination medicament according to any one of claims 80 - 82, wherein the target protein is a protein expressed on the cell surface of lymphocytes.

84. The combination medicament according to any one of claims 80 - 83, wherein the target protein is a subunit of a cytokine receptor interleukin - 2 (IL - 2) receptor, IL - 4 receptor, IL - 7 receptor, IL - 9 receptor, IL - 15 receptor or IL - 21 receptor.

85. The combination medicament according to any one of claims 80 - 84, wherein the target protein is interleukin - 2 receptor subunit gamma (IL2RG).

86. The combination medicament according to any one of claims 80 - 85, wherein the target protein antagonist selectively inhibits host cells in the subject based on the expression of the second subtype of the target protein by the host cells.

87. The combination medicament according to claim 86, wherein the selective inhibition of the host cells does not include ablating the host cells by an active killing mechanism.

88. The combination drug according to claim 86 or 87, wherein the selective inhibition comprises: (1) Blocking the growth of the host cells to provide a competitive growth advantage for the donor cells; (2) Blocking the localization or transport of the host cells to provide a competitive homing advantage for the donor cells; (3) Blocking the cell - to - cell interaction or adhesion of the host cells to provide a competitive tissue infiltration advantage for the donor cells; or (4) Blocking the activation of immune cells in the host cells to provide a competitive advantage for the donor cells.

89. The combination medicament according to any one of claims 86 - 88, wherein the selective inhibition includes blocking the growth of the host cells and / or blocking the activation of immune cells in the host cells to provide a competitive growth advantage for the donor cells.

90. The combination drug according to any one of claims 80 - 89, wherein the first subtype and the second subtype are functionally indistinguishable but immunologically distinguishable.

91. The combination drug according to any one of claims 80 - 90, wherein the donor cells express the first subtype of the target protein and the second subtype of the target protein.

92. The combination drug according to any one of claims 80 - 90, wherein the donor cells only express the first subtype of the target protein.

93. The combination drug according to any one of claims 80 - 92, wherein the first subtype of the target protein is expressed from an expression vector in the population of donor cells, or wherein the genomic locus has been edited to express the first subtype of the target protein in the population of donor cells.

94. The combination drug according to claim 93, wherein the genomic locus is an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is the IL2RG genomic locus.

95. The combination drug according to claim 93, wherein the genomic locus is not an endogenous genomic locus encoding the target protein, optionally, wherein the target protein is IL2RG, and the genomic locus is not the IL2RG genomic locus.

96. The combination drug according to any one of claims 80 - 95, wherein the first subtype of the target protein is a genetically engineered subtype of the target protein.

97. The combination drug according to any one of claims 80 - 96, wherein the first subtype of the target protein is genetically engineered to contain a mutation, thereby providing an altered epitope, optionally, wherein the mutation is an artificial mutation.

98. The combination drug according to claim 97, wherein the altered epitope is the binding region of an antagonist of the target protein, such that compared to its ability to bind and / or inhibit the second subtype of the target protein, the antagonist of the target protein exhibits reduced or eliminated ability to bind and / or inhibit the first subtype of the target protein.

99. The combination drug according to claim 98, wherein both the first subtype of the target protein and the second subtype of the target protein retain the ability to bind an endogenous ligand, optionally, wherein the antagonist of the target protein blocks the binding of the endogenous ligand to the second subtype of the target protein, but does not block the binding of the endogenous ligand to the first subtype of the target protein.

100. The combination drug according to any one of claims 97 - 99, wherein the target protein is IL2RG, wherein the altered epitope is in the binding region of the antagonist of the target protein, wherein the antagonist of the target protein is an antibody, which comprises an immunoglobulin light chain or its variable region, which comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and Wherein the three heavy chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NO: 4, 6, and 8.

101. The combination drug according to any one of claims 97-100, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus.

102. The combination drug according to any one of claims 97-101, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97.

103. The combination drug according to any one of claims 97-102, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147.

104. The combination drug according to any one of claims 97-103, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position M145. Optionally, the substitution is an M145K substitution, an M145D substitution, an M145E substitution, an M145P substitution, an M145W substitution, or an M145Y substitution.

105. The combination drug according to any one of claims 97-104, wherein the target protein is IL2RG, and wherein the mutation comprises an M145K substitution.

106. The combination drug according to any one of claims 97-105, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position W90. Optionally, the substitution is a W90V substitution, a W90R substitution, a W90Q substitution, a W90L substitution, a W90K substitution, a W90E substitution, or a W90D substitution. Optionally, the mutation comprises a W90Q substitution.

107. The combination drug according to any one of claims 97-106, wherein the target protein is IL2RG, and wherein the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

108. The combination drug according to any one of claims 80-107, wherein the target protein antagonist is an antigen-binding protein.

109. The combination drug according to claim 108, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

110. The combination drug according to claim 108 or 109, wherein the target protein is IL2RG, wherein the antigen-binding protein comprises an immunoglobulin light chain or its variable region, which comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy chain CDRs. wherein the three light-chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the same, and wherein the three heavy-chain CDRs each comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the same.

111. The combination drug according to claim 110, wherein the three light-chain CDRs each comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the same, and wherein the three heavy-chain CDRs each comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the same.

112. The combination drug according to claim 108 or 109, wherein the target protein is IL2RG, wherein the antigen-binding protein comprises an immunoglobulin light-chain variable region that comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the same, and wherein the antigen-binding protein comprises an immunoglobulin heavy-chain variable region that comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the same.

113. The combination drug according to claim 112, wherein the target protein is IL2RG, wherein the immunoglobulin light-chain variable region comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the same, and wherein the immunoglobulin heavy-chain variable region comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the same.

114. The combination drug according to claim 108 or 109, wherein the target protein is IL2RG, wherein the antigen-binding protein comprises an immunoglobulin light chain that comprises the sequence shown in SEQ ID NO: 20, consists essentially of or consists of the same, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain that comprises the sequence shown in SEQ ID NO: 18, consists essentially of or consists of the same.

115. The combination drug according to any one of claims 80-114, wherein the donor cell is a hematopoietic cell, optionally wherein the donor cell is an immune cell.

116. The combination drug according to any one of claims 80-115, wherein the donor cell is a lymphocyte or a lymphoid progenitor cell.

117. The combination drug according to any one of claims 80-116, wherein the donor cell is a T cell.

118. The combination drug according to any one of claims 80-117, wherein the donor cell is a tumor-infiltrating lymphocyte (TIL).

119. The combination drug according to any one of claims 80-116, wherein the donor cell is a B cell.

120. The combination drug according to any one of claims 80-116, wherein the donor cell is a NK cell.

121. The combination drug according to any one of claims 80 - 115, wherein the donor cell is a hematopoietic stem cell, or a hematopoietic stem cell and a progenitor cell.

122. The combination drug according to any one of claims 80 - 121, wherein the donor cell is derived from induced pluripotent stem cells, or from hematopoietic stem cells, or a hematopoietic stem cell and a progenitor cell.

123. The combination drug according to any one of claims 80 - 122, wherein the subject is a mammal or a non - mammalian mammal, and the donor cell is a mammalian cell or a non - mammalian mammalian cell.

124. The combination drug according to any one of claims 80 - 123, wherein the subject is human, and the donor cell is a human cell.

125. The combination drug according to any one of claims 80 - 124, wherein the donor cell contains or expresses a therapeutic molecule.

126. The combination drug according to claim 125, wherein the therapeutic molecule does not target the target protein.

127. The combination drug according to any one of claims 80 - 126, wherein the donor cell contains or expresses an immunoglobulin.

128. The combination drug according to claim 127, wherein the immunoglobulin does not target the target protein.

129. The combination drug according to any one of claims 80 - 128, wherein the donor cell contains a chimeric antigen receptor (CAR) or an exogenous T - cell receptor (TCR).

130. The combination drug according to claim 129, wherein the CAR or the exogenous TCR does not target the target protein.

131. The combination drug according to any one of claims 80 - 130, wherein the donor cell is autologous.

132. The combination drug according to any one of claims 80 - 130, wherein the donor cell is allogeneic or syngeneic.

133. The combination drug according to any one of claims 80 - 132, wherein the subject has a disease or disorder, and the combination drug is used to treat the disease or disorder.

134. The combination drug according to any one of claims 80 - 133, wherein the subject has cancer.

135. The combination drug according to claim 134, wherein the cancer is a solid tumor cancer.

136. The combination drug according to claim 134, wherein the cancer is a hematological cancer.

137. The combination drug according to any one of claims 80 - 132, wherein the subject has a hematopoietic malignancy, and the combination drug is used to treat the subject's hematopoietic malignancy.

138. The combination drug according to any one of claims 80 - 137, wherein the subject has defective immune cells or a hematopoietic genetic defect.

139. The combination drug according to claim 138, wherein the hematopoietic genetic defect is sickle cell disease or severe combined immunodeficiency (SCID).

140. An isolated cell or cell population that has been modified to express a first isoform of interleukin-2 receptor subunit gamma (IL2RG) that is different from a second isoform of IL2RG, wherein the first isoform of IL2RG has been genetically engineered to contain a mutation that provides an altered epitope, wherein the altered epitope is a binding region for an IL2RG antagonist such that the IL2RG antagonist exhibits reduced or eliminated ability to bind to and / or inhibit the first isoform of IL2RG as compared to its ability to bind to and / or inhibit the second isoform of IL2RG, and wherein the first isoform of IL2RG retains binding to its endogenous ligand.

141. The isolated cell or cell population of claim 140, wherein the mutation is an artificial mutation.

142. The isolated cell or cell population of claim 140 or 141, wherein both the first isoform of IL2RG and the second isoform of IL2RG retain the ability to bind to the endogenous ligand, optionally, wherein the IL2RG antagonist blocks binding of the endogenous ligand to the second isoform of IL2RG but does not block binding of the endogenous ligand to the first isoform of IL2RG.

143. The isolated cell or cell population of any one of claims 140-142, wherein the first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable.

144. The isolated cell or cell population of any one of claims 140-143, wherein the one or more cells express the first isoform of IL2RG and the second isoform of IL2RG.

145. The isolated cell or cell population of any one of claims 140-143, wherein the one or more cells express only the first isoform of IL2RG.

146. The isolated cell or cell population of any one of claims 140-145, wherein the first isoform of IL2RG is expressed in the one or more cells from an expression vector, or wherein the genomic locus has been edited to express the first isoform of IL2RG in the one or more cells.

147. The isolated cell or cell population of claim 146, wherein the genomic locus is the endogenous IL2RG genomic locus.

148. The isolated cell or cell population of claim 146, wherein the genomic locus is not the endogenous IL2RG genomic locus.

149. The isolated cell or cell population of any one of claims 140-148, wherein the altered epitope is in the binding region of the IL2RG antagonist, wherein the IL2RG antagonist is an antibody that comprises an immunoglobulin light chain or its variable region that comprises three light chain CDRs, and an immunoglobulin heavy chain or its variable region that comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO:12, 14, and 16, consist essentially of or consist of the same, and Wherein the three heavy chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NO: 4, 6, and 8.

150. The isolated cell or cell population according to any one of claims 140-149, wherein the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus.

151. The isolated cell or cell population according to any one of claims 140-150, wherein the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97.

152. The isolated cell or cell population according to any one of claims 140-151, wherein the mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147.

153. The isolated cell or cell population according to any one of claims 140-152, wherein the mutation comprises a mutation or substitution at position M145, optionally, wherein the substitution is M145K substitution, M145D substitution, M145E substitution, M145P substitution, M145W substitution, or M145Y substitution.

154. The isolated cell or cell population according to any one of claims 140-153, wherein the mutation comprises M145K substitution.

155. The isolated cell or cell population according to any one of claims 140-154, wherein the mutation comprises a mutation or substitution at position W90, optionally, wherein the substitution is W90V substitution, W90R substitution, W90Q substitution, W90L substitution, W90K substitution, W90E substitution, or W90D substitution, optionally, wherein the mutation comprises W90Q substitution.

156. The isolated cell or cell population according to any one of claims 140-155, wherein the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

157. The isolated cell or cell population according to any one of claims 140-156, wherein the first subtype of IL2RG and the second subtype of IL2RG can be immunologically distinguished by the IL2RG antagonist, wherein the IL2RG antagonist specifically binds to the second subtype of IL2RG but does not specifically bind to the first subtype of IL2RG.

158. The isolated cell or cell population according to any one of claims 140-157, wherein the IL2RG antagonist is an antigen-binding protein.

159. The isolated cell or cell population according to claim 158, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

160. The isolated cell or cell population according to claim 158 or 159, wherein the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the same, and wherein the three heavy chain CDRs respectively comprise a sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the same.

161. The isolated cell or cell population according to claim 160, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NOs: 12, 14, and 16, consists essentially of or consists of the same, and wherein the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NOs: 4, 6, and 8, consists essentially of or consists of the same.

162. The isolated cell or cell population according to claim 158 or 159, wherein the antigen-binding protein comprises an immunoglobulin light chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the same, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the same.

163. The isolated cell or cell population according to claim 162, wherein the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consists essentially of or consists of the same, and wherein the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consists essentially of or consists of the same.

164. The isolated cell or cell population according to claim 158 or 159, wherein the antigen-binding protein comprises an immunoglobulin light chain, which comprises the sequence shown in SEQ ID NO: 20, consists essentially of or consists of the same, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain, which comprises the sequence shown in SEQ ID NO: 18, consists essentially of or consists of the same.

165. The isolated cell or cell population according to any one of claims 140-164, wherein the one or more cells are hematopoietic cells, optionally wherein the one or more cells are immune cells.

166. The isolated cell or cell population according to any one of claims 140-165, wherein the one or more cells are lymphocytes or lymphoid progenitor cells.

167. The isolated cell or cell population according to any one of claims 140-166, wherein the one or more cells are T cells.

168. The isolated cell or cell population according to any one of claims 140 - 167, wherein the one or more cells are tumor infiltrating lymphocytes (TIL).

169. The isolated cell or cell population according to any one of claims 140 - 166, wherein the one or more cells are B cells.

170. The isolated cell or cell population according to any one of claims 140 - 166, wherein the one or more cells are NK cells.

171. The isolated cell or cell population according to any one of claims 140 - 165, wherein the one or more cells are hematopoietic stem cells, or hematopoietic stem cells and progenitor cells.

172. The isolated cell or cell population according to any one of claims 140 - 171, wherein the one or more cells are induced pluripotent stem cells.

173. The isolated cell or cell population according to any one of claims 140 - 172, wherein the one or more cells are mammalian cells or non - human mammalian cells.

174. The isolated cell or cell population according to any one of claims 140 - 173, wherein the one or more cells are human cells.

175. The isolated cell or cell population according to any one of claims 140 - 174, wherein the one or more cells contain or express a therapeutic molecule.

176. The isolated cell or cell population according to claim 175, wherein the therapeutic molecule does not target IL2RG.

177. The isolated cell or cell population according to any one of claims 140 - 176, wherein the one or more cells contain or express an immunoglobulin.

178. The isolated cell or cell population according to claim 177, wherein the immunoglobulin does not target IL2RG.

179. The isolated cell or cell population according to any one of claims 140 - 178, wherein the one or more cells contain a chimeric antigen receptor (CAR) or an exogenous T - cell receptor (TCR).

180. The isolated cell or cell population according to claim 179, wherein the CAR or the exogenous TCR does not target IL2RG.

181. The isolated cell or cell population according to any one of claims 140 - 180, wherein the one or more cells are isolated from a subject.

182. The isolated cell or cell population according to any one of claims 140 - 181, for treating a subject having cells expressing a second subtype of the IL2RG.

183. The isolated cell or cell population used according to claim 182, wherein the one or more cells are isolated from the subject.

184. A method of preparing the isolated cell or cell population according to any one of claims 140 - 183, comprising modifying a cell or cell population to express a first subtype of the IL2RG.

185. The method according to claim 184, wherein the modification comprises introducing an expression vector encoding the first subtype of the IL2RG, or wherein the modification comprises editing a genomic locus to express the first subtype of the IL2RG.

186. The method according to claim 185, wherein the genomic locus is the endogenous IL2RG genomic locus.

187. The method according to claim 185, wherein the genomic locus is not the endogenous IL2RG genomic locus.

188. The method according to any one of claims 185-187, wherein the editing comprises introducing the following into the cell: (1) a nuclease reagent or one or more nucleic acids encoding the nuclease reagent, wherein the nuclease reagent targets a nuclease target sequence in the genomic locus; and (2) an exogenous donor nucleic acid, wherein the nuclease reagent cleaves the genomic locus, and the exogenous donor nucleic acid inserts into or recombines with the genomic locus to produce the donor cell expressing the first subtype of IL2RG.

189. The method according to claim 188, wherein the nuclease reagent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein; and (ii) a guide RNA, wherein the guide RNA comprises a DNA targeting fragment that targets a guide RNA target sequence that is the nuclease target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

190. The method according to claim 189, wherein the nuclease reagent comprises the Cas protein and the guide RNA, optionally, wherein the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 76-87, or the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 64-75, or optionally, wherein the DNA targeting fragment comprises a sequence shown in any one of SEQ ID NOs: 136-153, or the guide RNA target sequence comprises a sequence shown in any one of SEQ ID NOs: 118-135.

191. The method according to claim 189 or 190, wherein the Cas protein is a Cas9 protein.

192. The method according to any one of claims 188-191, wherein the exogenous donor nucleic acid comprises homology arms.

193. The method according to any one of claims 188-192, wherein the exogenous donor nucleic acid is a single-stranded oligodeoxynucleotide (ssODN), optionally, wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 88-117, or optionally, wherein the ssODN comprises a nucleic acid sequence shown in any one of SEQ ID NOs: 154-173.

194. The method according to claim 193, wherein: (I) The DNA targeting fragment comprises the sequence shown in SEQ ID NO:77, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:65, and wherein the ssODN comprises the nucleic acid sequence shown in any one of SEQ ID NO:88 - 97; (II) The DNA targeting fragment comprises the sequence shown in SEQ ID NO:83, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:71, and wherein the ssODN comprises the nucleic acid sequence shown in any one of SEQ ID NO:98 - 107; (III) The DNA targeting fragment comprises the sequence shown in SEQ ID NO:86, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:74, and wherein the ssODN comprises the nucleic acid sequence shown in any one of SEQ ID NO:108 - 117; (IV) The DNA targeting fragment comprises the sequence shown in SEQ ID NO:137, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:119, and wherein the ssODN comprises the nucleic acid sequence shown in any one of SEQ ID NO:154 - 163; or (V) The DNA targeting fragment comprises the sequence shown in SEQ ID NO:138, or the guide RNA target sequence comprises the sequence shown in SEQ ID NO:120, and wherein the ssODN comprises the nucleic acid sequence shown in any one of SEQ ID NO:164 - 173.

195. A genetically engineered human interleukin - 2 receptor subunit gamma (IL2RG) protein, which comprises an artificial mutation to provide an altered epitope, wherein the altered epitope is a binding region of an IL2RG antagonist, such that compared with its ability to bind to and / or inhibit the wild - type human IL2RG protein, the IL2RG antagonist exhibits reduced or eliminated ability to bind to and / or inhibit the genetically engineered IL2RG protein, and wherein the genetically engineered IL2RG protein retains its binding to its endogenous ligand.

196. The genetically engineered human IL2RG protein according to claim 195, wherein the genetically engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable but immunologically distinguishable.

197. The genetically engineered human IL2RG protein according to claim 195 or 196, wherein the altered epitope is in the binding region of the IL2RG antagonist, wherein the IL2RG antagonist is an antibody, which comprises an immunoglobulin light chain or its variable region, which comprises three light - chain CDRs, and an immunoglobulin heavy chain or its variable region, which comprises three heavy - chain CDRs, wherein the three light - chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NO:12, 14, and 16, and Wherein the three heavy chain CDRs respectively comprise, consist essentially of, or consist of the sequences shown in SEQ ID NO: 4, 6, and 8.

198. The genetically engineered human IL2RG protein according to any one of claims 195-197, wherein the mutation comprises a mutation encoded by a nucleotide within exon 2 and / or exon 3 of the IL2RG gene at the IL2RG genomic locus.

199. The genetically engineered human IL2RG protein according to any one of claims 195-198, wherein the mutation comprises a mutation or substitution within the region from position T127 to position N150 and / or within the region from position L87 to position D97.

200. The genetically engineered human IL2RG protein according to any one of claims 195-199, wherein the artificial mutation comprises a mutation or substitution at position M145, position W90, position K92, position N93, position D95, position D97, position T127, position R139, position R140, position Q141, position T143, and / or position K147.

201. The genetically engineered human IL2RG protein according to any one of claims 195-200, wherein the artificial mutation comprises a mutation or substitution at position M145, optionally, wherein the substitution is M145K substitution, M145D substitution, M145E substitution, M145P substitution, M145W substitution, or M145Y substitution.

202. The genetically engineered human IL2RG protein according to any one of claims 195-201, wherein the artificial mutation comprises M145K substitution.

203. The genetically engineered human IL2RG protein according to any one of claims 195-202, wherein the mutation comprises a mutation or substitution at position W90, optionally, wherein the substitution is W90V substitution, W90R substitution, W90Q substitution, W90L substitution, W90K substitution, W90E substitution, or W90D substitution, optionally, wherein the mutation comprises W90Q substitution.

204. The genetically engineered human IL2RG protein according to any one of claims 195-203, wherein the mutation comprises a mutation or substitution at position M145 and a mutation or substitution at position W90.

205. The genetically engineered human IL2RG protein according to any one of claims 195-204, wherein the genetically engineered IL2RG protein and the native IL2RG protein are functionally indistinguishable, but can be immunologically distinguished by the IL2RG antagonist.

206. The genetically engineered human IL2RG protein according to any one of claims 195-205, wherein the IL2RG antagonist is an antigen-binding protein.

207. The genetically engineered human IL2RG protein according to claim 206, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

208. The genetically engineered human IL2RG protein according to claim 206 or 207, wherein the antigen-binding protein comprises an immunoglobulin light chain or a variable region thereof, which comprises three light chain CDRs, and an immunoglobulin heavy chain or a variable region thereof, which comprises three heavy chain CDRs, wherein the three light chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively comprise sequences that are at least 90% identical to the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same.

209. The genetically engineered human IL2RG protein according to claim 208, wherein the three light chain CDRs respectively comprise the sequences shown in SEQ ID NO: 12, 14, and 16, consist essentially of or consist of the same, and wherein the three heavy chain CDRs respectively comprise the sequences shown in SEQ ID NO: 4, 6, and 8, consist essentially of or consist of the same.

210. The genetically engineered human IL2RG protein according to claim 206 or 207, wherein the antigen-binding protein comprises an immunoglobulin light chain variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, consist essentially of or consist of the same, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain or variable region, which comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2, consist essentially of or consist of the same.

211. The genetically engineered human IL2RG protein according to claim 210, wherein the immunoglobulin light chain variable region comprises the sequence shown in SEQ ID NO: 10, consist essentially of or consist of the same, and wherein the immunoglobulin heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, consist essentially of or consist of the same.

212. The genetically engineered human IL2RG protein according to claim 206 or 207, wherein the antigen-binding protein comprises an immunoglobulin light chain, which comprises the sequence shown in SEQ ID NO: 20, consist essentially of or consist of the same, and wherein the antigen-binding protein comprises an immunoglobulin heavy chain, which comprises the sequence shown in SEQ ID NO: 18, consist essentially of or consist of the same.

213. A nucleic acid encoding the genetically engineered human IL2RG protein according to any one of claims 195 - 212, optionally, wherein the nucleic acid is an expression vector encoding the genetically engineered human IL2RG protein.

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