Cells engineered with HLA-E and HLA-G transgenes

Abstract

The present disclosure provides genetically engineered cells and their derivatives, in particular cells and their derivatives modified with HLA-E and HLA-G transgenes.Related vectors, nuclease complexes, polypeptides, polynucleotides, and pharmaceutical compositions are also provided.Methods for treating subjects using genetically engineered cells and / or pharmaceutical compositions are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 338,329, filed May 4, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. Said XML copy, created on May 4, 2023, is named 256805_000030_SL.xml and is 210,228 bytes in size.

[0003] Field The present disclosure provides genetically engineered cells and their derivatives, in particular cells and their derivatives modified with HLA-E and HLA-G transgenes.Related vectors, nuclease complexes, polypeptides, polynucleotides, and pharmaceutical compositions are also provided.Methods for treating subjects using genetically engineered cells and / or pharmaceutical compositions are also provided. [Background technology]

[0004] Autologous, patient-specific, immunotherapy has emerged as a powerful and potentially curative treatment. However, autologous immune cells must be generated on a custom-made basis, which remains a significant limiting factor for large-scale clinical application due to production costs and the risk of production failure. Moreover, for cancer patients, especially those who have undergone multiple rounds of chemotherapy and drug treatment before being eligible for immunotherapy, the quality and count of the cells to be engineered may be low. Furthermore, there is a risk of contamination of the final therapeutic composition by malignant cells. At the same time, the high variability of the composition makes it difficult to maintain critical quality attributes, which may reduce the safety and efficacy of the treatment.

[0005] Allogeneic immunotherapy (i.e., using cells from a non-patient donor) has advantages over autoimmunotherapy that make it an attractive option. In many cases, allogeneic immunotherapy relies on "off the shelf" products, meaning that the patient receives cells originating from a healthy donor that have been genetically engineered to elicit the required therapeutic response. These allogeneic immunotherapy compositions will contain consistent batches that can be stored and shipped to the patient as needed. Thus, the patient receives immunotherapy on demand, which saves valuable time and resources. Although allogeneic immunotherapy has many advantages, it still has major challenges to overcome. Two of the biggest challenges involve cytokine release syndrome (CRS), in which the patient's immune cells are activated by the donor cells, releasing large amounts of cytokines into the body, and graft-versus-host disease (GvHD), in which the patient's T cells attack the donor cells, causing a life-threatening reaction.

[0006] Therefore, there is an unmet need for therapeutically sufficient and functional allogeneic immune cells for effective use in immunotherapy.

[0007] Furthermore, in cell engineering therapeutics, it is desirable to minimize the number of gene edits that need to be made to the cells. Thus, there is a need for engineered cell therapies that have multiple functionalities that can be engineered with a minimal number of edits. Summary of the Invention

[0008] The present invention describes compositions and methods for use in the genomic engineering of cells, such as induced pluripotent stem cells (iPSCs). Specifically, the methods and compositions described relate to compositions and methods for introducing HLA-E and HLA-G transgenes into iPSCs, such as pluripotent hematopoietic stem and / or progenitor cells (HSC / PCs), and for preparing immune effector cells derived from iPSCs.

[0009] Polymorphisms in human leukocyte antigen (HLA) class I and class II genes can cause rejection of iPSC-derived products in allogeneic recipients. Disruption of the beta-2 microglobulin (B2M) gene can eliminate the surface expression of all class I molecules, and disruption of the CIITA gene can eliminate the expression of all class II molecules. However, since HLA class I molecules function as inhibitory ligands for NK cells, cells that do not present HLA class I molecules are attacked and killed by NK cells. Thus, disruption of HLA I genes leaves cells susceptible to lysis by natural killer (NK) cells. This "loss of self" response can be prevented by forced expression of minimally polymorphic HLA-E and HLA-G molecules. Therefore, gene editing can be utilized to knock-in the HLA-E and / or HLA-G genes in human iPSCs to confer inducible and regulated surface expression of HLA-E and / or HLA-G single chain dimers. See, for example, Nat Biotechnol. 2017 Aug; 35(8): 765-772. By doing this, these HLA-engineered iPSCs and their differentiated derivatives are resistant to NK-mediated lysis. Furthermore, the inhibition of NK-mediated lysis can be enhanced by combining both HLA-E and HLA-G. By combining HLA-E and HLA-G components in a single transgene according to the present invention, the number of gene edits that need to be made to the cells is minimized. In addition, by combining HLA-E and HLA-G components in one construct, it may result in less expression of one or the other coding sequence, which may limit function. Therefore, the linkage between the HLA-E and / or HLA-G components needs to be optimized to achieve the desired expression.

[0010] In one aspect, the invention provides a chimeric single chain HLA-E and HLA-G molecule comprising: (a) a first molecule comprising an HLA-E heavy chain and (b) a second molecule comprising an HLA-G heavy chain, and (c) a connecting peptide between (a) and (b).

[0011] In some embodiments, the order of the chimeric single chain HLA-E and HLA-G molecules can be (i)(a)-(c)-(b) or (ii)(b)-(c)-(a).

[0012] In some embodiments, the HLA-E heavy chain polypeptide may comprise the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto.

[0013] In some embodiments, the nucleotide sequence encoding the HLA-E heavy chain polypeptide may comprise the sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 80% sequence identity thereto.

[0014] In some embodiments, the HLA-G heavy chain polypeptide may comprise the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 80% sequence identity thereto.

[0015] In some embodiments, the nucleotide sequence encoding the HLA-G heavy chain polypeptide may comprise the sequence of SEQ ID NO:26, or a nucleotide sequence having at least 80% sequence identity thereto.

[0016] In some embodiments, the connecting peptide may comprise an autoprotease peptide and optionally one or two autoprotease peptide linkers.

[0017] In some embodiments, at least one of the autoprotease peptide linkers can be 5' to the autoprotease peptide, 3' to the autoprotease peptide, or both 5' and 3' to the autoprotease peptide.

[0018] In some embodiments, the autoprotease peptide can comprise an amino acid sequence set forth in Table 3, or an amino acid sequence having at least 80% sequence identity thereto.

[0019] In some embodiments, the autoprotease peptide can be a 2A peptide.

[0020] In some embodiments of any of the various chimeric single chain HLA-E and HLA-G molecules disclosed herein, the 2A peptide can be a P2A, F2A, E2A, T2A, GF2A, GP2A, GE2A, GT2A, BmCPV2A, or BmIFV2A peptide.

[0021] In some embodiments, the 2A peptide can be a P2A peptide.

[0022] In some embodiments, the P2A peptide may comprise the amino acid sequence of SEQ ID NO:21, or an amino acid sequence having at least 80% sequence identity thereto.

[0023] In some embodiments, the nucleotide sequence encoding the P2A peptide may comprise the sequence of SEQ ID NO:22, or a nucleotide sequence having at least 80% sequence identity thereto.

[0024] In some embodiments of any of the various chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) a first molecule may comprise a first B2M polypeptide fused to an HLA-E heavy chain via a first linker, and / or (b) a second molecule may comprise a second B2M polypeptide fused to an HLA-G heavy chain via a second linker.

[0025] In some embodiments, the first B2M polypeptide may be 5' to an HLA-E heavy chain polypeptide.

[0026] In some embodiments, the first B2M polypeptide may be 3' to an HLA-E heavy chain polypeptide.

[0027] In some embodiments, the second B2M polypeptide may be 5' to the HLA-G heavy chain polypeptide.

[0028] In some embodiments, the second B2M polypeptide may be 3' to the HLA-G heavy chain polypeptide.

[0029] In some embodiments, the first B2M polypeptide and / or the second B2M polypeptide may comprise the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 80% sequence identity thereto.

[0030] In some embodiments, the polynucleotide sequence encoding the first B2M polypeptide and / or the second B2M polypeptide may comprise the sequence of SEQ ID NO: 10 or 11, or a nucleotide sequence having at least 80% sequence identity thereto.

[0031] In some embodiments of any of the various chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first molecule may further comprise a first presenting peptide fused to the first B2M polypeptide via a third linker, and / or (b) the second molecule may further comprise a second presenting peptide fused to the second B2M polypeptide via a fourth linker.

[0032] In some embodiments of any of the various chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first presenting peptide may be fused to a first B2M polypeptide, and (a) the second presenting peptide may be fused to a second B2M polypeptide.

[0033] In some embodiments, the first and / or second presented peptides can be the same.

[0034] In some embodiments, the first and / or second presented peptides may be different.

[0035] In some embodiments, the first and / or second presentation peptide can comprise the amino acid sequence of SEQ ID NO: 4 or 23, or an amino acid sequence having at least 80% sequence identity thereto.

[0036] In some embodiments, the polynucleotide sequence encoding the first and / or second presentation peptide may comprise the sequence of SEQ ID NO: 5 or 24, or a nucleotide sequence having at least 80% sequence identity thereto.

[0037] In some embodiments, the first, second, third, fourth, and / or autoprotease peptide linkers may each separately comprise an amino acid sequence set forth in Table 4, or an amino acid sequence having at least 80% sequence identity thereto.

[0038] In some embodiments, the first peptide linker sequence and / or the second peptide linker sequence may comprise the amino acid sequence of SEQ ID NO: 6, 39 or 41, or an amino acid sequence having at least 80% sequence identity thereto.

[0039] In some embodiments, the nucleotide sequence encoding the first peptide linker sequence and / or the second peptide linker sequence may comprise SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 80% sequence identity thereto.

[0040] In some embodiments, the third peptide linker sequence and / or the fourth peptide linker sequence may comprise the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto.

[0041] In some embodiments, the nucleotide sequence encoding the third peptide linker sequence and / or the fourth peptide linker sequence may comprise the sequence of SEQ ID NO: 13 or 14, or a nucleotide sequence having at least 80% sequence identity thereto.

[0042] In some embodiments of any of the various chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first molecule may further comprise a first signal peptide operably linked to the HLA-E heavy chain, and / or (b) the second molecule may further comprise a second signal peptide operably linked to the HLA-G heavy chain.

[0043] In some embodiments, the first signal peptide and the second signal peptide can be the same.

[0044] In some embodiments, the first signal peptide and the second signal peptide can be different.

[0045] In some embodiments, the first signal peptide and / or the second signal peptide may comprise the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity thereto.

[0046] In some embodiments, the first signal peptide and the second signal peptide may comprise the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity thereto.

[0047] In some embodiments, the polynucleotide sequence encoding the first signal peptide and / or the second signal peptide may comprise the sequence of SEQ ID NO: 2 or 3, or a polynucleotide sequence having at least 80% sequence identity thereto.

[0048] In some embodiments, the first molecule may comprise the amino acid sequence of SEQ ID NO: 17 or 19, or an amino acid sequence having at least 80% sequence identity thereto.

[0049] In some embodiments, the second molecule may comprise the amino acid sequence of SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity thereto.

[0050] In some embodiments, the chimeric single chain HLA-E and HLA-G molecules of the present disclosure may comprise the amino acid sequence of SEQ ID NO:31, 165 or 168.

[0051] In another aspect, the invention provides polynucleotides encoding any of the various single chain HLA-E and HLA-G molecules disclosed herein.

[0052] In some embodiments, the polynucleotide sequence encoding the first molecule may comprise the nucleotide sequence of SEQ ID NO: 18 or 20, or a nucleotide sequence having at least 80% sequence identity thereto.

[0053] In some embodiments, the polynucleotide sequence encoding the second molecule may comprise the nucleotide sequence of SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity thereto.

[0054] In some embodiments, a polynucleotide encoding the single chain HLA-E and HLA-G molecules of the present disclosure may comprise the nucleotide sequence of SEQ ID NO:32, 120, 166, 167 or 169.

[0055] In some embodiments, the polynucleotide sequences encoding the single chain HLA-E and HLA-G molecules of the present disclosure may be operably linked to a single promoter.

[0056] In some embodiments, the promoter can be an inducible promoter.

[0057] In some embodiments of the above polynucleotides, the polynucleotide may be a DNA molecule.

[0058] In some embodiments of the above polynucleotides, the polynucleotide may be an RNA molecule.

[0059] In another aspect, the present invention provides recombinant vectors comprising any of the various polynucleotides disclosed herein.

[0060] In some embodiments, the vector may be a viral vector.

[0061] In some embodiments, the viral vector can be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a herpes viral vector, a baculoviral vector, or a vaccinia viral vector.

[0062] In some embodiments, the vector may be a non-viral vector.

[0063] In some embodiments, the non-viral vector can be a minicircle plasmid, a Sleeping Beauty transposon, a piggyBac transposon, or a single- or double-stranded DNA molecule used as a template for homology-directed repair (HDR)-based gene editing.

[0064] In another aspect, the present invention provides an isolated cell comprising any of the various polynucleotides disclosed herein or any of the various recombinant vectors disclosed herein.

[0065] In another aspect, the present invention provides an isolated host cell comprising any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein encoded by any of the various polynucleotides disclosed herein.

[0066] In some embodiments, the host cell may be an iPSC or a population thereof.

[0067] In some embodiments, the host cell may be an immune effector cell.

[0068] In another aspect, the present invention provides immune effector cells or populations thereof derived from any of the various iPSCs disclosed herein.

[0069] In some embodiments of the isolated host cells, immune effector cells, or populations disclosed herein, the host cells, immune effector cells, or populations thereof may be T cells, natural killer (NK) cells, natural killer T cells (NKT cells), mesenchymal stem cells (MSCs), or macrophages.

[0070] In some embodiments, the host cell, immune effector cell, or population thereof may be a T cell.

[0071] In some embodiments, the host cell, immune effector cell, or population thereof may be an αβ T cell receptor (TCR) T cell, a γδ T cell, a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an invariant natural killer T (iNKT) cell, a memory T cell, a memory T stem cell (TSCM), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

[0072] In some embodiments, the host cells, immune effector cells, or populations thereof may be NK cells.

[0073] In another aspect, the present invention provides a MAD7 / gRNA ribonucleoprotein (RNP) complex composition for insertion of HLA-E and HLA-G transgenes, the MAD7 / gRNA ribonucleoprotein (RNP) complex composition comprising: (I) MAD7 nuclease; (II) a guide RNA (gRNA) specific for MAD7 nuclease, wherein the gRNA may comprise a guide sequence capable of hybridizing to a target sequence of an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, the guide sequence being selected from SEQ ID NOs: 109-119, and wherein when the gRNA is complexed with the MAD7 nuclease, a gRNA specific for MAD7 nuclease, the guide sequence directing sequence-specific binding of MAD7 nuclease to the target sequence; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to the left and right arms of a target sequence in the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus, (2) a promoter, (3) a polynucleotide encoding an HLA-E and HLA-G transgene comprising any of the various polynucleotides disclosed herein, to which the promoter is operably linked, and (4) a transcription terminator sequence.

[0074] In another aspect, the present invention provides a MAD7 / gRNA ribonucleoprotein (RNP) complex composition for insertion of HLA-E and HLA-G transgenes, the MAD7 / gRNA ribonucleoprotein (RNP) complex composition comprising I) a MAD7 nuclease system encoded by one or more vectors, the one or more vectors comprising (a) a sequence encoding a guide RNA (gRNA) operably linked to a first regulatory element, the gRNA may comprise a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38 or CLYBL locus in a cell, the guide sequence being selected from SEQ ID NOs: 109-119, and wherein when transcribed, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence. and (II) an HLA-E and HLA-G transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of a target sequence in an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL locus; (2) a promoter; (3) a polynucleotide encoding an HLA-E and HLA-G transgene comprising any of the various polynucleotides disclosed herein, operably linked to the promoter; and (4) a transcription terminator sequence.

[0075] In another aspect, the present invention provides a MAD7 / gRNA ribonucleoprotein (RNP)-based vector system, the MAD7 / gRNA ribonucleoprotein (RNP)-based vector system comprising: (I) one or more vectors, comprising: (a) a sequence encoding a guide RNA (gRNA), the sequence being operably linked to a first regulatory element, the gRNA may comprise a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL locus in a cell, the gRNA guide sequence being selected from SEQ ID NOs: 109-119, and wherein when transcribed, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; (b) a sequence encoding a guide RNA (gRNA), the sequence being operably linked to a first regulatory element, the gRNA may comprise a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL locus in a cell, the gRNA guide sequence being selected from SEQ ID NOs: 109-119, and wherein when transcribed, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; and (II) one or more vectors comprising a sequence encoding MAD7 nuclease, the sequence being operably linked to a second regulatory element; and (II) HLA-E and HLA-G transgene vectors comprising (1) left and right polynucleotide sequences homologous to left and right arms of a target sequence in the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus, (2) a promoter, (3) a polynucleotide encoding an HLA-E and HLA-G transgene comprising any of the various polynucleotides disclosed herein, to which the promoter is operably linked, and (4) a transcription terminator sequence.

[0076] In some embodiments of the above compositions or the above vector systems, the cells may be induced pluripotent stem cells (iPSCs).

[0077] In some embodiments, the first and / or second regulatory element may be a promoter.

[0078] In some embodiments, the first regulatory element and the second regulatory element can be the same.

[0079] In some embodiments, the first regulatory element and the second regulatory element may be different.

[0080] In some embodiments, the gRNA guide sequence may be specific for the AAVS1 locus.

[0081] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO: 109.

[0082] In some embodiments, the gRNA guide sequence may be specific for the B2M locus.

[0083] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO:110.

[0084] In some embodiments, the gRNA guide sequence may be specific for the CIITA locus.

[0085] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO: 111 or 112.

[0086] In some embodiments, the gRNA guide sequence may be specific for the NKG2A locus.

[0087] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO:114.

[0088] In some embodiments, the gRNA guide sequence may be specific for the TRAC locus.

[0089] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO:115.

[0090] In some embodiments, the gRNA guide sequence may be specific for the CD70 locus.

[0091] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO:116.

[0092] In some embodiments, the gRNA guide sequence may be specific for the CD38 locus.

[0093] In some embodiments, the gRNA guide sequence may comprise SEQ ID NO:117.

[0094] In some embodiments, the gRNA guide sequence may be specific for the CD33 locus.

[0095] In some embodiments, the gRNA guide sequence may be specific for the CD33 locus and may include SEQ ID NO: 118 or 119.

[0096] In some embodiments, the gRNA guide sequence may be specific for the CLYBL locus.

[0097] In some embodiments, the gRNA guide may include SEQ ID NO:113.

[0098] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of AAVS1 may comprise the nucleotide sequences of SEQ ID NOs: 73 and 74, respectively, or fragments thereof.

[0099] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of B2M may comprise the nucleotide sequences of SEQ ID NOs: 76 and 77, respectively, or fragments thereof.

[0100] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CIITA may comprise (i) the nucleotide sequences of SEQ ID NOs: 79 and 80, respectively, or (ii) SEQ ID NOs: 95 and 96, respectively, or fragments thereof.

[0101] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of NKG2A may comprise the nucleotide sequences of SEQ ID NOs: 85 and 86, respectively, or fragments thereof.

[0102] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of TRAC may comprise the nucleotide sequences of SEQ ID NOs: 88 and 89, respectively, or fragments thereof.

[0103] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CD70 may comprise the nucleotide sequences of SEQ ID NOs: 98 and 99, respectively, or fragments thereof.

[0104] In some embodiments, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CLYBL may comprise the nucleotide sequences of SEQ ID NOs: 82 and 83, respectively, or fragments thereof.

[0105] In some embodiments, when the RNP complex is introduced into a cell, expression of an endogenous gene comprising a target sequence complementary to the guide sequence of the gRNA molecule can be reduced or eliminated in said cell.

[0106] In another aspect, the present invention provides one or more retroviruses that make up any of the various vector systems disclosed herein.

[0107] In another aspect, the present invention provides an isolated host cell transformed by any of the various vector systems disclosed herein or any of the various retroviruses disclosed herein.

[0108] In some embodiments, the host cell may be an iPSC or a population thereof.

[0109] In some embodiments, the host cell may be an immune effector cell.

[0110] In another aspect, the present invention provides immune effector cells or populations thereof derived from any of the iPSCs disclosed herein.

[0111] In some embodiments, the host cell, immune effector cell, or population thereof may be a T cell, a natural killer (NK) cell, a natural killer T cell (NKT cell), a mesenchymal stem cell (MSC), or a macrophage.

[0112] In some embodiments, the host cell, immune effector cell, or population thereof may be a T cell.

[0113] In some embodiments, the host cell, immune effector cell, or population thereof may be an αβ T cell receptor (TCR) T cell, a γδ T cell, a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an invariant natural killer T (iNKT) cell, a memory T cell, a memory T stem cell (TSCM), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

[0114] In some embodiments, the host cell may be a NK cell.

[0115] In another aspect, the invention provides pharmaceutical compositions comprising any of the various isolated host cells or iPSC-derived immune effector cells disclosed herein.

[0116] In another aspect, the present invention provides a method for preventing or treating cancer comprising administering to an individual in need thereof a therapeutically effective amount of any of the various host cells, immune effectors and / or populations disclosed herein, or any of the various pharmaceutical compositions disclosed herein.

[0117] In some embodiments, the cancer may be selected from the group consisting of lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer.

[0118] In some embodiments, the cancer may be selected from the group consisting of leukemia, e.g., AML, CML, ALL, and CLL, lymphoma, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, and solid cancer, e.g., sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and brain cancer, and cancer of unknown etiology (CUP).

[0119] In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment.

[0120] In some embodiments, an individual may have no minimal residual disease (MRD) after one or more cancer treatments or repeat administrations.

[0121] In some embodiments, the isolated host cells, immune effector cells, or populations thereof disclosed herein exert improved protection against allogeneic cell lysis compared to cells that do not express the chimeric single chain HLA-E and HLA-G molecules of the present disclosure.

[0122] In some embodiments, the present application provides a method of protecting immune effector cells from allogeneic cell lysis, comprising introducing into the immune effector cells a polynucleotide encoding a chimeric single-chain HLA-E and HLA-G molecule described herein, or a recombinant vector thereof, or a MAD7 / gRNA ribonucleoprotein (RNP) complex composition described herein, or a MAD7 / gRNA ribonucleoprotein (RNP)-based vector system described herein. In some embodiments, the immune effector cells are T cells, natural killer (NK) cells, natural killer T cells (NKT cells), mesenchymal stem cells (MSCs), or macrophages. In some embodiments, the immune effector cells are T cells, e.g., αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, memory T stem cells (T SCM ), naive T cells, effector T cells, T helper cells, or regulatory T cells (Tregs). In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are derived from iPSCs. [Brief description of the drawings]

[0123] [Figure 1] FIG. 1 depicts an exemplary schematic diagram of HLA-E and HLA-G transgenes. The diagram discloses "(G4S)4" as in SEQ ID NO:39, and "(G4S)3" as in SEQ ID NO:6. [Diagram 2] FIG. 1 depicts the AAVS1 targeting vector map. [Diagram 3] FIG. 1 depicts a B2M targeting vector map. [Figure 4] FIG. 1 depicts the CIITA targeting vector map. [Diagram 5] FIG. 1 depicts the CLYBL targeting vector map. [Figure 6] FIG. 1 depicts the NKG2A targeting vector map. [Figure 7] FIG. 1 depicts the TRAC targeting vector map. [Figure 8] FIG. 1 depicts the CIITA targeting vector map. [Figure 9] FIG. 1 depicts a map of the CD70 targeting vector. [Figure 10] FIG. 1 depicts flow cytometry analysis of engineered cells showing expression of both HLA-E (e.g., see top panel) and HLA-G (e.g., see bottom panel) after homologous recombination repair (HDR) in induced pluripotent stem cells (iPSCs) to iPSCs. [Figure 11] FIG. 1 depicts flow cytometry analysis of both HLA-E and HLA-G expression of engineered cells engineered by HDR into iPSCs and enriched for HLA-E expression only. [Figure 12-1] 12A-12B: Depicts exemplary HLA-E and HLA-G transgene amino acid sequences (FIG. 12A) and corresponding nucleic acid sequences (FIG. 12B). [Figure 12-2] (As stated above.) [Figure 13-1] Figures 13A-13B: HLA-E expression on K562 cells provided improved protection from killing over HLA-G, while the combination of both HLA-E and HLA-G conferred improved protection against peripheral blood mononuclear cell (PBMC) cytolysis. Figure 13A shows a cytotoxicity assay using engineered K562 cells expressing HLA-G, HLA-E, HLA-E and HLA-G or parental WT control K562 cells as control cells and PBMCs derived from healthy donors as effector cells. Cells were co-cultured for 72 hours at multiple effector to target ratios (E:T). The dashed straight lines show representative calculated IC50 values ​​(shown in the legend of Figure 13B). Figure 13B shows the specific cytolysis calculated by dividing the IC50 value of the parental control by the IC50 value of the engineered K562 line. Each data point represents a different individual PBMC donor. [Figure 13-2] (As stated above.) [Figure 14-1]14A-14B: iNK cells edited with HLA-E and HLA-G were consistently protective compared to iNK cells lacking HLA. Cytotoxicity assays using B2M KO iNK or B2M KO iNK with HLA-E and HLA-G using PBMCs derived from healthy donors as effector cells. Cells were co-cultured for 72 hours at multiple effector to target ratios (E:T). Dashed lines show representative calculated IC50 values ​​(shown in legend to FIG. 14B) (FIG. 14A). Specific cell lysis calculated by dividing the IC50 value of B2M KO iNK cells by the IC50 value of B2M KO iNK cells with HLA-E and HLA-G. Each data point represents a different individual PBMC donor (FIG. 14B). [Figure 14-2] (As stated above.) [Figure 15-1] Figures 15A-15D: iPSCs edited with HLA-E and HLA-G linked by different peptide linkers are expressed in iPSCs. Figures 15A-15B show HLA-E expression, and Figures 15C-15D show HLA-G expression. [Figure 15-2] (As stated above.) [Figure 15-3] (As stated above.) [Figure 15-4] (As stated above.) [Figure 16-1] Figures 16A-16B: Comparison of HLA-E (Figure 16A) and HLA-G (Figure 16B) expression in iPSCs edited with HLA-E and HLA-G linked by different peptide linkers. [Figure 16-2] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0124] The present application provides, among other things, HLA-E and HLA-G transgenes, compositions and methods for use in genome engineering of cells such as iPSCs. Specifically, the methods and compositions described relate to introducing nucleic acids encoding HLA-E and HLA-G transgenes into iPSCs, such as pluripotent hematopoietic stem and / or progenitor cells (HSC / PCs), and preparing immune effector cells derived from iPSCs, such as T cells, NK cells, macrophages and dendritic cells. Specifically, DNA sequences encoding chimeric single-chain HLA-E and HLA-G molecules, gene transfer vectors for genome engineering of human cell lines, and methods of their use are disclosed. The gene transfer vectors are designed to insert transgenes into the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, and / or CLYBL loci of human cells (iPSCs), and include promoter sequences, terminator sequences and homology arms specific to the loci of interest. The gene transfer vector can be used with CRISPR nuclease-based system, for example, MAD7 nuclease-based system.The guide sequence is also included for use with the CRISPR nuclease-based system for inserting transgene, particularly with the MAD7 nuclease-based system.In some embodiments, the MAD7 nuclease-based system comprises non-naturally occurring or engineered MAD7 nuclease.

[0125] I. Definition Various publications, articles, and patents are cited or described in the background and throughout this specification. Each of these references is incorporated herein by reference in its entirety for all intended purposes. Discussion of documents, acts, materials, devices, articles, or the like included in this specification is for the purpose of providing a context for the embodiments of the present disclosure. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Otherwise, certain terms used herein have the meanings set forth herein.

[0127] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0128] Unless otherwise indicated, the term "at least" preceding a series of elements is to be construed to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Such equivalents are intended to be encompassed by the present application.

[0129] As used herein, the terms "comprises," "comprising," "includes," "including," "having," "having," "containing," or "containing" or any other variations thereof shall be interpreted to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or absent); A is false (or absent) and B is true (or present); and A and B are both true (or present).

[0130] As used herein, the conjunctive term "and / or" between multiple stated elements is interpreted as including both individual and combined options. For example, when two elements are conjoined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is included in the meaning and thus is interpreted as meeting the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one of the options is also included in the meaning and thus is interpreted as meeting the requirements of the term "and / or".

[0131] As used herein, the term "consists of" or variations such as "consist of" or "consisting of" as used throughout this specification and claims indicates the inclusion of any stated integer or group of integers, but indicates that additional integers or groups of integers cannot be added to the specified method, structure, or composition.

[0132] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" as used throughout the specification and claims indicates the inclusion, by necessity, of any stated integer or group of integers that does not materially change the basic or novel characteristics of the method, structure, or composition being stated. See the United States Patent Examining Manual, Chapter 2111.03.

[0133] As used herein, "subject" means any animal, preferably a mammal, most preferably a human. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like, more preferably humans. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual.

[0134] It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to dimensions or characteristics (e.g., concentrations or concentration ranges) of components of the present invention indicate that the described dimensions / characteristics are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. Unless otherwise stated, any numerical values, such as concentrations or concentration ranges described herein, should be interpreted as being modified in all instances by the term "about." At a minimum, such references involving numerical parameters will include variations that do not alter the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.). In some embodiments, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges includes all the possible subranges, and all individual numerical values ​​within that range, including integers and fractions of values ​​within such ranges, unless the context clearly dictates otherwise.

[0135] The term "identical" or percent "identity" in the context of two or more nucleic acids (e.g., guide RNA sequences or homologous arm sequences) or polypeptide sequences (e.g., chimeric single chain HLA-E and HLA-G molecules) refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below or by visual inspection.

[0136] For sequence comparison, typically, one sequence serves as the reference sequence with which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on designated program parameters.

[0137] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)).

[0138] Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0139] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by X amount from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program for amino acid sequences uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0140] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which is an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001 in the comparison of the test nucleic acid and the reference nucleic acid.

[0141] Another indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is usually substantially identical to a second polypeptide, for example, when the two peptides are only different in terms of conservative substitution.Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0142] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid, a peptide, a protein, or a cell) has been substantially separated, produced at a location, or purified from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues, of the organism in which the component naturally occurs. Thus, "isolated" nucleic acids, peptides, proteins, and cells include nucleic acids, peptides, proteins, and cells purified by standard purification methods and the purification methods described herein. "Isolated" nucleic acids, peptides, proteins, and cells may be part of a composition, and still be said to be isolated when the composition is not part of the native environment of the nucleic acid, peptide, protein, and cell. The term also encompasses chemically synthesized nucleic acids, as well as nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell.

[0143] As used herein, the term "polynucleotide", synonymously referred to as "nucleic acid molecule", "nucleotide" or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA; DNA that is a mixture of single-stranded and double-stranded regions; single-stranded and double-stranded RNA; and RNA that is a mixture of single-stranded and double-stranded regions; and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions, including RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA that contains one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases, and unconventional bases, such as inosine. A variety of modifications can be made to DNA and RNA, and thus "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms of polynucleotides that are typically found in nature, as well as the chemical forms of DNA and RNA that are characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short nucleic acid strands that are often referred to as "oligonucleotides."

[0144] "Construct" refers to a macromolecule or complex of molecules, including a polynucleotide, that is delivered to a host cell, either in vitro or in vivo. "Vector" as used herein refers to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell where it can replicate and / or express. The term "vector" as used herein includes the construct to be delivered. A vector can be a linear or circular molecule. A vector can be integrative or non-integrative. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and Sendai viral vectors.

[0145] "Integration" or "insertion" means that one or more sequences or nucleotides of an exogenous construct are stably inserted into a cell genome, i.e., covalently linked to a nucleic acid sequence in a chromosome or mitochondrial DNA of a cell. "Targeted integration" means that the nucleotides of a construct are inserted into a preselected site or "integration site" in a chromosome or mitochondrial DNA of a cell. The term "integration" or "insertion" as used herein further refers to a process that includes the insertion of one or more sequences or nucleotides of an exogenous construct, with or without the deletion of an endogenous sequence or one or more nucleotides at the integration site. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted endogenous sequence or one or more nucleotides with one or more inserted sequences or nucleotides.

[0146] As used herein, the term "exogenous" is intended to mean that the referenced molecule or the referenced activity is introduced into or non-native to the host cell. The molecule may be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material, for example, by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in reference to the expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid in an expressible form into the cell. The term "endogenous" refers to the referenced molecule or activity that is present in the host cell in its native form. Similarly, when used in reference to the expression of an encoding nucleic acid, the term "endogenous" refers to the expression of an encoding nucleic acid that is natively contained within the cell and not exogenously introduced.

[0147] As used herein, a "transgene", "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences in vivo, is transcribed into RNA and, in some cases, translated into a polypeptide. A gene or polynucleotide of interest can include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest can code for miRNA, shRNA, a native polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a variant of a native polypeptide that has less than 100% sequence identity with the native peptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, and a selectable marker, etc.

[0148] "Operably linked" refers to the operably linking of nucleic acid or amino acid sequences such that they are placed in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in a sense or antisense orientation.

[0149] The term "expression" as used herein refers to the biosynthesis of gene products. This term includes the transcription of genes into RNA. This term also includes the translation of RNA into one or more polypeptides, and further includes any naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide (e.g., single-chain HLA-E and HLA-G molecule polypeptides and / or CAR polypeptides) can be in the cytoplasm of host cells, can enter the extracellular environment, e.g., the growth medium of cell culture, or can be anchored to the cell membrane.

[0150] The term "regulatory element" refers to any cis-acting genetic element that controls some aspect of the expression of a nucleic acid sequence. In some embodiments, the term "promoter" essentially includes the minimum sequence required to initiate transcription. In some embodiments, the term "promoter" includes a sequence for initiating transcription, and also includes a sequence that can upregulate or downregulate transcription, which are commonly referred to as "enhancer elements" and "repressor elements", respectively.

[0151] In some embodiments, the term "promoter" essentially includes the minimal sequence required to initiate transcription. In some embodiments, the term "promoter" includes sequences for initiating transcription, and also includes sequences that can upregulate or downregulate transcription, which are commonly referred to as "enhancer elements" and "repressor elements," respectively.

[0152] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter designations for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can include modified amino acids, and can be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, for example, by conjugation with a labeling component. Also included within this definition are polypeptides that contain one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0153] Peptide sequences described herein are written according to the conventional convention with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomers of amino acids are known, the L-form of an amino acid is shown unless expressly indicated otherwise.

[0154] II. Induced pluripotent stem cells (iPSCs) and immune effector cells Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs, are a type of pluripotent stem cells that are artificially derived from non-pluripotent cells, typically adult somatic cells, by inserting certain genes.Induced pluripotent stem cells are considered to be identical to natural pluripotent stem cells, such as embryonic stem cells, in many aspects, for example, in the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and ability and differentiation potential, although the full relationship between induced pluripotent stem cells and natural pluripotent stem cells is still being evaluated.iPS cells were first produced from mouse cells in 2006 (Takahashi et al., 2006) and from human cells in 2007 (Takahashi et al., 2007; Yu et al, 2007). This has been heralded as a significant advancement in stem cell research, as it has enabled researchers to obtain pluripotent stem cells, which are important in research and could potentially be used therapeutically, without the controversial use of embryos.

[0155] Without wishing to be bound by theory, human iPSC technology represents a promising and potentially limitless source of therapeutically effective hematopoietic cells for the treatment of numerous hematologic and non-hematologic malignancies, including cancer. To advance the bright prospects of human iPSCs and genetically engineered iPSC technology as an allogeneic source of hematopoietic cell therapy, it is desirable to be able to generate both efficiently and reproducibly not only hematopoietic stem and progenitor cells (HSCs), but also immune effector populations, including, for example, diverse subsets of T, B, NKT, and NK lymphoid cells with desired genetic modifications, as well as their progenitors. Therefore, methods and complexes are needed for efficiently inserting genetic elements into human iPSCs for therapeutic use.

[0156] iPSCs have unlimited self-renewal potential. Their use allows for the manipulation of cells to produce controlled cell banks of modified cells that can be expanded and differentiated into desired immune effector cells, thereby providing large quantities of homogenous allogeneic therapeutic products.

[0157] Provided herein are genetically engineered iPSCs and their derived cells. Provided herein are selected genomic modifications that enhance the therapeutic properties of derived cells. Derived cells are functionally improved and suitable for allogeneic ready-made cell therapy, receiving a combination of selected modalities that are introduced into cells at the iPSC level by genomic engineering. This approach can help reduce the side effects mediated by cytokine release syndrome CRS / GVHD and prevent autoimmunity for a long period of time while providing excellent efficacy.

[0158] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells. Specialized cells include, for example, blood cells or muscle cells. Differentiated or differentiation-induced cells are cells that have taken a more specialized ("committed") position in the lineage of a cell. The term "committed" when applied to the process of differentiation refers to a cell that has progressed in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or a subset of cell types, and cannot differentiate into a different cell type or revert to a less differentiated cell type under normal circumstances. As used herein, the term "pluripotency" refers to the ability of a cell to form all the lineages of the body or somatic cells or germ spheres. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells that cannot give rise to an entire organism (e.g., epiblast stem cells or EpiSCs), to more primitive, more pluripotent cells that can give rise to an entire organism (e.g., embryonic stem cells).

[0159] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells produced from differentiated adult, neonatal or fetal cells that are induced or changed or reprogrammed into cells that can differentiate into tissues of all three germ layers or layers: ectoderm, mesoderm and endoderm. The produced iPSCs do not refer to the cells as they are found in nature.

[0160] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but have the potential for further hematopoietic differentiation. Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (blood cell progenitors), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T cells, B cells, NK cells).

[0161] As used herein, the term "immune cell" or "immune effector cell" refers to a cell that participates in immune response. Immune response includes, for example, promoting immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and bone marrow-derived macrophages. In some embodiments, immune effector cells can be, for example, T cells, natural killer (NK) cells, natural killer T cells (NKT cells), mesenchymal stem cells (MSCs), or macrophages, or populations thereof.

[0162] As used herein, the term "engineered immune cells" or "engineered immune effector cells" refers to immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell. As used herein, the terms "T lymphocytes" and "T cells" are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and plays various roles in the immune system. T cells can play roles, including, for example, identifying specific foreign antigens in the body, and activating and inactivating other immune cells. T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells harvested from a mammal. T cells can be CD3+ cells. The T cells may be any type of T cell and may be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, and gamma delta T cells (gdT cells; γδT cells). Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). T cells may also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells may also be differentiated from stem or progenitor cells.

[0163] "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by a secretory profile following stimulation that may include secretion of cytokines, such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55 kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associative recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, they define helper / inducer subsets.

[0164] "CD8+ T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the associative recognition element in major histocompatibility complex class I restricted interactions.

[0165] As used herein, the term "NK cell" or "natural killer cell" is a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptor (CD3). NK cell can also refer to genetically engineered NK cell, for example, NK cell modified to express chimeric antigen receptor (CAR). NK cell can also be differentiated from stem cell or progenitor cell.

[0166] Induced pluripotent stem cell (iPSC) parent cell lines can be generated from peripheral blood mononuclear cells (PBMCs) or T cells using any known method for introducing reprogramming factors into non-pluripotent cells, for example, an episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140, ​​9,644,184, 9,328,332 and 8,765,470, the complete disclosures of which are incorporated herein by reference in their entirety for all intended purposes. Reprogramming factors can be in the form of polynucleotides and are thus introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by lentiviral vectors. In some embodiments, one or more polynucleotides are introduced by episomal vectors. In various other embodiments, one or more polynucleotides are introduced by Sendai virus vectors. In some embodiments, the iPSCs are clonal iPSCs or are obtained from a pool of iPSCs, and genome editing is introduced by one or more targeted integrations and / or in / dels at one or more selected sites. In another embodiment, the iPSCs are obtained from human T cells with antigen specificity and rearranged TCR genes (hereinafter also referred to as "T-iPS" cells), as described in U.S. Patent Nos. 9,206,394 and 10,787,642, which are hereby incorporated by reference in their entirety for all intended purposes.

[0167] III. Derived immune effector cells In another aspect, the present disclosure relates to derived immune effector cells, which are derived cells from the differentiation of iPSC.As mentioned above, the genome editing introduced into iPSC is retained in derived immune effector cells.In certain embodiments of derived cells obtained from iPSC differentiation, the derived cells are hematopoietic cells, including but not limited to HSC (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells.In certain embodiments, the derived cells are immune effector cells, such as NK cells or T cells.

[0168] In certain embodiments, the present application provides natural killer (NK) cells or T cells derived from iPSCs with one or more transgene inserts prepared according to the present disclosure.

[0169] A method for producing a derivative cell is also provided, the method comprising the step of differentiating the iPSCs under conditions for cell differentiation, thereby obtaining a derivative cell.

[0170] The iPSC of the present application can be differentiated by any method known in the art.Exemplary methods are described in U.S. Patent No. 8,846,395, U.S. Patent No. 8,945,922, U.S. Patent No. 8,318,491, and International Patent Publication No. WO 2010 / 099539, WO 2012 / 109208, WO 2017 / 070333, WO 2017 / 179720, WO 2016 / 010148, WO 2018 / 048828 and WO 2019 / 157597, each of which is incorporated herein by reference in its entirety for all intended purposes.

[0171] IV. Targeted genome editing at selected loci in iPSCs According to embodiments of the present application, one or more of the exogenous polynucleotides are inserted into one or more loci on one or more chromosomes of the iPSCs.

[0172] Genome editing or genomic editing or gene editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted and / or replaced in the genome of a targeted cell. Targeted genome editing (synonymous with "targeted genome editing" or "targeted gene editing") allows insertion, deletion and / or replacement at a preselected site of the genome. If an endogenous sequence is deleted or destroyed at the insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or knocked down due to sequence deletion or destruction. Therefore, targeted editing can also be used to precisely disrupt endogenous gene expression. The terms "targeted integration" and "targeted insertion" are used interchangeably herein and refer to a process that involves the insertion of one or more exogenous sequences into a preselected site of the genome, with or without the deletion of the endogenous sequence at the insertion site.

[0173] Various methods and compositions have been described for targeted cleavage of genomic DNA. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, to induce targeted deletion of cellular DNA sequences, and to facilitate targeted recombination at a given chromosomal locus. These methods often involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks in target DNA sequences so that the repair of the cleavage by error-prone processes such as non-homologous end joining (NHEJ) or repair using a repair template (homologous recombination repair or HDR) can result in knockout of a gene or insertion of a sequence of interest (targeted integration). Cleavage can be performed by using specific nucleases, such as engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR / Cas systems, together with engineered crRNA / tracr RNA ("single guide RNA") to guide specific cleavage.

[0174] Targeted editing can be achieved by either nuclease-independent or nuclease-dependent approaches. In nuclease-independent targeted editing approaches, homologous recombination is guided by the host cell's enzymatic machinery through homologous sequences flanking the inserted exogenous polynucleotide.

[0175] Alternatively, targeted editing can be achieved more frequently by specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertion or deletion (in / del) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homologous arms, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration".

[0176] Targeted nucleases include naturally occurring and recombinant nucleases, such as CRISPR-associated nucleases from families including Cas, Cpf, Cse, Csy, Csn, Csd, Cst, Csh, Csa, Csm and Cmr; restriction endonucleases; meganucleases; and homing endonucleases. As an example, CRISPR / Cpf1 includes two main components: (1) Cpf1 endonuclease, and (2) guide nucleic acid, which can be DNA or RNA. When co-expressed, the two components form a ribonucleoprotein (RNP) complex, which is recruited to a target DNA sequence that includes a PAM and a seed region near the PAM. The guide nucleic acid can be used to guide Cpf1 to target a selected sequence. These two components can then be delivered to mammalian cells by transfection or transduction.

[0177] Cpf1 (also known as Cas12a), a member of the alternative CRISPR nuclease family, has been used for genome editing since its first report in 2015 (Zetsche et al Cell, 163(3), 759-771). Cpf1 nuclease exhibits distinct features from Cas9 nuclease, such as staggered DSBs, T-rich PAMs, and the native use of only one guide RNA molecule to form a complex with Cpf1 and target DNA. These features allow Cpf1 nuclease to be used in target organisms or in regions within the genome of organisms where the use of Cas9 is less feasible due to lower GC content.

[0178] Recently, an alternative CRISPR nuclease called MAD7 was disclosed in U.S. Patent Nos. 9,982,279 and 10,337,028, the contents of which are hereby incorporated in their entirety for all intended purposes. Inscripta has made this nuclease free for any commercial or academic research. Its use for commercial genome editing has therefore attracted great interest. Inscripta reports that MAD7 was developed from Eubacterium rectale and has demonstrated its functionality in E. coli, S. cerevisiae and in the human HEK293T cell line. MAD7 shares only 31% identity with Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which also shares a T-rich PAM site (5'-YTTN-3') and a 21-nucleotide protospacer (region of the gRNA that associates the nuclease with the DNA target) length. Certain embodiments of the present disclosure are particularly suitable for use with the endonuclease MAD7. This nuclease requires only the crRNA for gene editing and allows for specific targeting of AT-rich regions of the genome. MAD7 cleaves DNA by staggered cuts compared to S. pyogenes, which has a blunt cut.

[0179] Exemplary MAD7 sequences and scaffold sequences of guide nucleic acids are provided in Table 1. In general, a "scaffold sequence" includes any sequence having a sequence sufficient to promote the formation of a targetable ribonucleoprotein complex. A targetable ribonucleoprotein complex may include a nucleic acid-guided nuclease (e.g., MAD7) and a guide nucleic acid including a scaffold sequence and a guide sequence. A sequence sufficient to promote the formation of a targetable ribonucleoprotein complex within a scaffold sequence may include a degree of complementarity along the length of two sequence regions within the scaffold sequence, such as one or two sequence regions involved in the formation of a secondary structure (e.g., a pseudoknot region). The one or two sequence regions may be included or encoded on the same polynucleotide. Alternatively, the one or two sequence regions may be included or encoded on separate polynucleotides. In some embodiments, the scaffold sequence may include any one of the sequences of SEQ ID NOs: 106-108. In some embodiments, the scaffold sequence includes the sequence of SEQ ID NO: 106. In some embodiments, the scaffold sequence includes the sequence of SEQ ID NO: 107. In some embodiments, the scaffold sequence comprises the sequence of SEQ ID NO:108.

[0180] [Table 1-1]

[0181] [Table 1-2]

[0182] [Table 1-3]

[0183] [Table 1-4]

[0184] [Table 1-5]

[0185] Thus, one aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genomic insertion utilizing MAD7 endonuclease. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired insertion site, and the targeted insertion method comprises introducing the construct into a cell to allow site-specific homologous recombination by a cellular host enzyme machinery. In another embodiment, the targeted insertion method into a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a CRISPR MAD7 expression cassette comprising a DNA binding domain specific to a desired insertion site into the cell. Specifically, according to the present disclosure, the targeted insertion method into a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell for insertion into a specific locus in iPSCs by introducing MAD7 nuclease and a gRNA comprising a guide sequence specific to a desired insertion site into the cell to allow MAD7-mediated insertion.

[0186] Generally, guide nucleic acid can be complexed with compatible nucleic acid guided nuclease and hybridize with target sequence, thus directing nuclease to target sequence.Guide nucleic acid can be DNA.Guide nucleic acid can be RNA.Guide nucleic acid can include both DNA and RNA.Guide nucleic acid can include modified or non-natural nucleotides.When guide nucleic acid includes RNA, RNA guide nucleic acid can be encoded by DNA sequence on polynucleotide molecule such as plasmid, linear construct or editing cassette as disclosed herein. In particular, in certain embodiments of the present disclosure, the guide sequence is for use with a MAD7 / gRNA ribonucleoprotein (RNP) complex for insertion of a transgene into a specific locus of an iPSC, the MAD7 / gRNA ribonucleoprotein (RNP) complex comprising: (I) a guide RNA (gRNA) polynucleotide sequence specific for MAD7 nuclease, the polynucleotide sequence being capable of hybridizing to a safe harbor locus in iPSCs (e.g., an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus) and capable of hybridizing to a guide RNA ( ... safe harbor locus in iPSCs (e.g., an AAVS1, B2M, CIITA, (II) a MAD7 enzyme protein; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to the left and right arms of a target sequence of a safe harbor locus (e.g., AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus), (2) a promoter, (3) a polynucleotide encoding a transgene of interest to which the promoter is operably linked, and (4) a transcription terminator sequence. In one embodiment, the guide sequence comprises a nucleotide sequence selected from SEQ ID NOs: 109-119.

[0187] The site for targeted insertion includes, but is not limited to, genomic safe harbor, which is an intragenic or extragenic region of the human genome that can theoretically accommodate the predictable expression of newly inserted DNA without adverse effects on host cell or organism.In certain embodiments, the genomic safe harbor for targeted insertion is one or more loci selected from the group consisting of AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL locus.

[0188] In other embodiments, the site for targeted insertion is selected to eliminate or reduce the expression of endogenous genes at the insertion site.As used herein, the term "deletion" in relation to gene expression refers to any genetic modification that eliminates the expression of genes.Examples of the "deletion" of gene expression include, for example, the removal or deletion of the DNA sequence of a gene, the insertion of an exogenous polynucleotide sequence into the locus of a gene, and one or more substitutions within a gene, which eliminate the expression of a gene.

[0189] Genes for targeted deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and class II proteins. Multiple MHC class I and class II proteins must be matched to histocompatibility in allogeneic recipients to avoid allogeneic rejection problems. "MHC-deficient", including MHC class I-deficient or MHC class II-deficient, or both, refers to cells that lack, no longer maintain, or have reduced levels of surface expression of complete MHC complexes, including MHC class I protein heterodimers and / or MHC class II heterodimers, such that the reduced or reduced levels are lower than the levels that can be naturally detected by other cells or by synthetic methods. MHC class I deficiency can be achieved by the deletion of the function of any region of the MHC class I locus (chromosome 6 p21) or the deletion or reduction of the expression level of one or more MHC class I related genes, including but not limited to the beta-2 microglobulin (B2M) gene, the TAP 1 gene, the TAP 2 gene and the tapasin gene. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers. B2M null cells are MHC-I deficient cells. MHC class II deficiency can be achieved by the deletion or reduction of the function of MHC-II related genes, including but not limited to RFXANK, CIITA, RFX5 and RFXAP. CIITA is a transcriptional coactivator that functions by activating the transcription factor RFX5, which is required for class II protein expression. CIITA null cells are MHC-II deficient cells. In certain embodiments, one or more of the exogenous polynucleotides are inserted into one or more loci of a gene selected from the group consisting of B2M, TAP 1, TAP 2, tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, thereby deleting or reducing expression of the inserted gene.

[0190] In certain embodiments, exogenous polynucleotide is inserted into one or more loci on the chromosome of cell.In certain embodiments, one or more loci are selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAPI, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, CD70, CD38, CD33 or TIGIT gene loci, with the proviso that at least one of the one or more loci is selected from the group consisting of B2M, TAP 1, TAP 2, tapasin, RFXANK, CIITA, RFX5 and RFXAP gene loci of MHC gene. In certain embodiments, one or more exogenous polynucleotides are inserted into the locus of MHC class I-related genes, such as beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or tapasin gene; and into the locus of MHC-II-related genes, such as RFXANK, CIITA, RFX5, RFXAP or CIITA gene; and optionally into the locus of a safe harbor gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR and RUNX1 genes. In certain embodiments, one or more exogenous polynucleotides are inserted into the locus of CIITA, AAVS1 and B2M genes.

[0191] In certain embodiments, multiple transgenes can be inserted into the site targeted for deletion of complex (MHC) class I and MHC class II proteins.For example, (a) a first exogenous polynucleotide can be inserted into the locus of AAVS1 gene, (b) a second exogenous polypeptide can be inserted into the locus of CIITA gene, and a third exogenous polypeptide can be inserted into the locus of B2M gene, and the insertion of exogenous polynucleotides deletes or reduces the expression of CIITA and B2M genes.

[0192] In certain embodiments, the guide RNA for insertion into the AAVS1 locus comprises a guide sequence of SEQ ID NO: 109 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 73 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 74 or a fragment thereof.

[0193] In certain embodiments, the guide RNA for insertion into the B2M locus comprises a guide sequence of SEQ ID NO: 110 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 76 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 77 or a fragment thereof.

[0194] In certain embodiments, the guide RNA for insertion into the CIITA locus comprises a guide sequence of SEQ ID NO: 111 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 79 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 80 or a fragment thereof. In certain embodiments, the guide RNA for insertion into the CIITA locus comprises a guide sequence of SEQ ID NO: 112 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 95 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 96 or a fragment thereof.

[0195] In certain embodiments, the guide RNA for insertion into the NKG2A locus comprises a guide sequence of SEQ ID NO: 114 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 85 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 86 or a fragment thereof.

[0196] In certain embodiments, the guide RNA for insertion into the TRAC locus comprises a guide sequence of SEQ ID NO: 115 or a variant thereof, the left homology arm comprises a nucleotide sequence of SEQ ID NO: 88 or a fragment thereof, and the right homology arm comprises a nucleotide sequence of SEQ ID NO: 89 or a fragment thereof.

[0197] In certain embodiments, the guide RNA for insertion into the CLYBL locus comprises a guide sequence of SEQ ID NO: 113 or a variant thereof, the left homologous arm comprises a nucleotide sequence of SEQ ID NO: 82 or a fragment thereof, and the right homologous sequence is selected from SEQ ID NO: 83 or a fragment thereof.

[0198] In certain embodiments, the guide RNA for insertion into the CD70 locus comprises a guide sequence of SEQ ID NO: 116 or a variant thereof, the left homologous arm comprises a nucleotide sequence of SEQ ID NO: 98 or a fragment thereof, and the right homologous sequence is selected from SEQ ID NO: 99 or a fragment thereof.

[0199] In certain embodiments, the guide RNA for insertion into the CD38 locus comprises the guide sequence of SEQ ID NO: 117 or a variant thereof.

[0200] In certain embodiments, the guide RNA for insertion into the CD33 locus comprises the guide sequence of SEQ ID NO: 118 or 119, or a variant thereof.

[0201] Targeting domain sequences (both RNA and DNA sequences are provided) and corresponding homology arm sequences of gRNA molecules for use in the compositions and methods of the disclosure, for example, in altering expression of or modifying iPSC target genes, are provided in Table 2.

[0202] [Table 2-1]

[0203] [Table 2-2]

[0204] V. Homology Arms Whether single-stranded or double-stranded, the donor template generally contains one or more regions that are homologous to a region of DNA, e.g., the target nucleic acid, within or near (e.g., adjacent or contiguous with) the target sequence to be cleaved, e.g., the cleavage site. These regions of homology are referred to herein as "homology arms" and are shown diagrammatically below: [5' homology arm]-[replacement sequence]-[3' homology arm].

[0205] The homologous arm of the donor template described herein can be of any suitable length, provided that such length is sufficient to allow efficient resolution of the cut site on the nucleic acid targeted by the DNA repair process that requires the donor template.In certain embodiments, if the amplification of the homologous arm is desired, for example by PCR, the homologous arm is of such a length that the amplification can be performed.In certain embodiments, if the sequencing of the homologous arm is desired, the homologous arm is of such a length that the sequencing can be performed.In certain embodiments, if the quantitative evaluation of amplicons is desired, the homologous arm is of such a length that the similar amplification number of each amplicon is achieved, for example, by similar G / C content, amplification temperature, etc.In certain embodiments, the homologous arm is double-stranded.In certain embodiments, the double-stranded homologous arm is single-stranded.

[0206] In certain embodiments, the 5' homology arm is between 50 and 250 nucleotides in length. In certain embodiments, the 5' homology arm is about 50, 75, 100, 125, 150, 175, 200, 225, or 250 nucleotides in length.

[0207] In certain embodiments, the 3' homology arm is between 50 and 250 nucleotides in length. In certain embodiments, the 3' homology arm is about 50, 75, 100, 125, 150, 175, 200, 225, or 250 nucleotides in length.

[0208] The 5' and 3' homology arms may be of the same length or may be different in length. In certain embodiments, the 5' and 3' homology arms are amplified to allow quantitative evaluation of gene editing events, such as targeted insertion, in the target nucleic acid. In certain embodiments, quantitative evaluation of gene editing events may rely on the amplification of both the 5' and 3' junctions at the site of targeted insertion by amplifying the whole or part of the homology arms using a single PCR primer pair in a single amplification reaction. Thus, even if the 5' and 3' homology arms may be different in length, it should be possible to amplify (e.g., using PCR) the length of each homology arm as desired. Furthermore, if it is desired to amplify both the 5' and 5' homology arms and the difference in length of the 3' homology arms in a single PCR reaction, the difference in length of the 5' and 3' homology arms should allow PCR amplification using a single PCR primer pair.

[0209] VI. HLA-E and HLA-G molecules The present application provides, among other things, HLA-E and HLA-G transgenes, compositions and methods for use in the genomic engineering of cells such as iPSCs.

[0210] HLA-G (HGNC:4964; NCBI Entrez Gene:3135; Ensembl:ENSG00000204632; OMIM®:142871; UniProtKB / Swiss-Prot:P17693) and HLA-E (HGNC:4962; NCBI Entrez Gene:3133; Ensembl:ENSG00000204592; OMIM®:143010; UniProtKB / Swiss-Prot:P13747) are members of the HLA class Ib family, known for their involvement in immune regulatory processes, particularly at the maternal-fetal interface and in immune self-non-self discrimination, respectively. Under allogeneic conditions, such as allotransplantation or pregnancy, HLA-G expression is associated with the increased tolerance of allografts or fetuses. Therefore, HLA-G is highly involved in immune tolerance. As an example, from a mechanistic point of view, short-term tolerance can be achieved by HLA-G through the interaction of allograft-derived β2-associated HLA-G1 and / or HLA-G5 molecules with Ig-like transcription factor 2 (ILT2) receptors on T, NK and B cells, resulting in inhibition of proliferation, cytotoxicity or antibody production. Short-term tolerance can also be indirectly elicited by HLA-G, for example, through the presentation of HLA-G-specific leader peptides by HLA-E and their interaction with the inhibitory receptor CD94 / NKG2A on T and NK cells, thus inhibiting NK cell lysis against cells expressing normal levels of HLA class I molecules. Interaction of HLA-G5 with the CD8 co-receptor on certain T and NK cell populations can lead to the deletion of these cells. Long-term tolerance can be achieved by the induction of different types of regulatory T (Treg) cells. Taken together, HLA-G and HLA-E may contribute to enhanced survival of donor cells, e.g., MHC-mismatched donor cells, as universal donor cells capable of inducing "stealth" immune tolerance (i.e., evasion of immune responses), which is associated with reduced susceptibility to immune rejection.

[0211] In certain aspects, the present disclosure provides chimeric single-chain HLA-E and HLA-G molecules. In some embodiments, the chimeric single-chain HLA-E and HLA-G molecules of the present disclosure may comprise (a) a first molecule comprising an HLA-E heavy chain and (b) a second molecule comprising an HLA-G heavy chain, and (c) a linking peptide between (a) and (b). In some embodiments, the order of the chimeric single-chain HLA-E and HLA-G molecules is (i) (a)-(c)-(b) or (ii) (b)-(c)-(a).

[0212] In some embodiments, the HLA-E heavy chain polypeptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15, or in certain embodiments, the amino acid sequence of SEQ ID NO: 15. In some embodiments, the HLA-E heavy chain polypeptide comprises an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the HLA-E heavy chain polypeptide comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the HLA-E heavy chain polypeptide comprises the sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 80% sequence identity thereto.

[0213] In some embodiments, the HLA-G heavy chain polypeptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25, or in certain embodiments, the amino acid sequence of SEQ ID NO: 25. In some embodiments, the HLA-G heavy chain polypeptide comprises an amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the HLA-G heavy chain polypeptide comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 26, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the nucleotide sequence encoding the HLA-G heavy chain polypeptide comprises the sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 80% sequence identity thereto.

[0214] In certain embodiments, the HLA-E and / or HLA-G heavy chains comprise mutated transmembrane domains. A transmembrane domain is a portion of a transmembrane protein (e.g., an HLA-E and / or HLA-G molecule) that spans across the cell membrane and anchors the molecule to the cell membrane. In some embodiments, the transmembrane domain can be modified by amino acid substitution, deletion, or insertion to minimize interaction with other members of the HLA-E and HLA-G molecules. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution, deletion, or insertion to avoid binding of proteins naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain comprises additional amino acids to allow flexibility and / or optimal distance between domains connected to the transmembrane domain.

[0215] In some embodiments, the HLA-E and / or HLA-G heavy chain polypeptides disclosed herein comprise fragments of an HLA-E and / or HLA-G heavy chain.

[0216] In certain embodiments, the HLA-E heavy chain fragment comprises an α1, α2, and / or a3 domain. In certain embodiments, the HLA-E heavy chain fragment comprises at least an α1 domain. In certain embodiments, the HLA-E heavy chain fragment comprises at least an α2 domain. In certain embodiments, the HLA-E heavy chain fragment comprises at least an α3 domain. In certain embodiments, the HLA-E heavy chain fragment comprises an α1 and an α2 domain. In certain embodiments, the HLA-E heavy chain fragment comprises an α1, α2, and a3 domain. In certain embodiments, the HLA-G heavy chain fragment comprises an α1, α2, and / or a3 domain. In certain embodiments, the HLA-G heavy chain fragment comprises at least an α1 domain. In certain embodiments, the HLA-G heavy chain fragment comprises at least an α2 domain. In certain embodiments, the HLA-G heavy chain fragment comprises at least an α3 domain. In certain embodiments, the HLA-G heavy chain fragment comprises an α1 and an α2 domain. In certain embodiments, the HLA-G heavy chain fragment comprises the α1, α2, and a3 domains.

[0217] In certain embodiments, the HLA-E heavy chain fragment and / or the HLA-G heavy chain fragment comprises a heterologous transmembrane domain.The transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0218] In some embodiments, the transmembrane domain may be derived from the HLA-E heavy chain polypeptide disclosed herein. In some embodiments, the transmembrane domain may be derived from the HLA-G heavy chain polypeptide disclosed herein. It will be understood by those skilled in the art that the transmembrane domain derived from the HLA-E and / or HLA-G heavy chain polypeptide disclosed herein can be replaced with other transmembrane domains that are not derived from HLA-E or HLA-G heavy chain polypeptides. In some embodiments, the transmembrane domain derived from the HLA-E and / or HLA-G heavy chain polypeptide disclosed herein can be replaced with any number of different transmembrane domains known in the art.

[0219] Non-limiting examples of transmembrane domains that may be particularly useful in the present disclosure may be derived from (i.e., at least include) the α, β or ζ chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain primarily hydrophobic residues, such as leucine and valine. For example, triplets of phenylalanine, tryptophan and / or valine may be found at each end of the synthetic transmembrane domain.

[0220] In some embodiments, the transmembrane domain may be derived from CD8α, CD28, CD8, CD4, CD3ζ, CD40, CD134 (OX-40), NKG2A / C / D / E, or CD7. In some embodiments, the transmembrane domain may be derived from CD28.

[0221] In certain embodiments, it may be desirable to utilize transmembrane domains of the zeta, eta, or FcεR1 gamma chains that contain cysteine ​​residues capable of disulfide bonding, so that the resulting chimeric protein can form disulfide-linked dimers with itself or with unmodified versions of the zeta, eta, or FcεR1 gamma chains or related proteins. In some cases, the transmembrane domains will be selected or modified by amino acid substitutions to avoid binding of the domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it may be desirable to utilize transmembrane domains of zeta, eta, or FcεR1 gamma and -β, MB1 (Igα.), B29, or CD3-γ, zeta, or eta, to retain physical association with other members of the receptor complex.

[0222] In some embodiments, the transmembrane domain is derived from an HLA-E heavy chain. In one embodiment, the HLA-E transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 161, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 161.

[0223] In some embodiments, the transmembrane domain is derived from an HLA-G heavy chain. In one embodiment, the HLA-G transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 162, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 162.

[0224] In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 154, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 154.

[0225] In some embodiments, the transmembrane domain is derived from CD8α. In one embodiment, the CD8α transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 159, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 159.

[0226] In some embodiments, the transmembrane domain is derived from CD28. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 155, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 155. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 158, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 158.

[0227] In some embodiments, the transmembrane domain is derived from CD3zeta. In one embodiment, the CD3zeta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 160, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 160.

[0228] In some embodiments, the transmembrane domain that may be particularly useful in the present disclosure may be an epidermal growth factor receptor (EGFR) transmembrane domain, or a fragment or derivative thereof. In some embodiments, the transmembrane domain is derived from EGFR. In one embodiment, the EGFR transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 163, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 163.

[0229] In some embodiments of any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein, the connecting peptide comprises an autoprotease peptide. Examples of autoprotease peptides include, but are not limited to, any of the peptide sequences shown in Table 3, or combinations thereof. Non-limiting examples of autoprotease peptides that can be used according to the present disclosure are listed in Table 3.

[0230] [Table 3]

[0231] In some embodiments, the autoprotease peptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set out in Table 3. In some embodiments, the autoprotease peptide comprises an amino acid sequence set out in Table 3, or an amino acid sequence having at least 80% sequence identity thereto.

[0232] In some embodiments, the autoprotease peptide is a 2A peptide. Non-limiting examples of 2A peptides include P2A, F2A, E2A, T2A, GF2A, GP2A, GE2A, GT2A, BmCPV2A, or BmIFV2A peptides.

[0233] In some embodiments, the 2A peptide is a P2A peptide.

[0234] In some embodiments, the P2A peptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 21, or in certain embodiments, the amino acid sequence of SEQ ID NO: 21. In some embodiments, the P2A peptide comprises an amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the P2A peptide comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding the P2A peptide comprises the sequence of SEQ ID NO: 22, or a nucleotide sequence having at least 80% sequence identity thereto.

[0235] In some embodiments, the connecting peptide comprises an autoprotease peptide and optionally an autoprotease peptide linker. In some embodiments, the connecting peptide comprises an autoprotease peptide and optionally two autoprotease peptide linkers.

[0236] In certain embodiments of any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first molecule may comprise a first B2M polypeptide fused to an HLA-E heavy chain via a first linker, and / or (b) the second molecule may comprise a second B2M polypeptide fused to an HLA-G heavy chain via a second linker. In certain embodiments of any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first molecule may comprise a first B2M polypeptide fused to an HLA-E heavy chain via a first linker, and (b) the second molecule may comprise a second B2M polypeptide fused to an HLA-G heavy chain via a second linker.

[0237] In some embodiments, the first B2M polypeptide may be 5' to the HLA-E heavy chain polypeptide. In some embodiments, the first B2M polypeptide may be 3' to the HLA-E heavy chain polypeptide. In some embodiments, the second B2M polypeptide may be 5' to the HLA-G heavy chain polypeptide. In some embodiments, the second B2M polypeptide may be 3' to the HLA-G heavy chain polypeptide. In some embodiments, the first B2M polypeptide may be 5' to the HLA-E heavy chain polypeptide and the second B2M polypeptide may be 5' to the HLA-G heavy chain polypeptide.

[0238] In some embodiments, the first and / or second B2M polypeptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9, or in certain embodiments, the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first and / or second B2M polypeptide comprises an amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the first and / or second B2M polypeptide comprises a nucleotide sequence that is at least 80%, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10 or 11, or in certain embodiments the nucleotide sequence of SEQ ID NO: 10 or 11. In some embodiments, the nucleotide sequence encoding the first and / or second B2M polypeptide comprises the sequence of SEQ ID NO: 10 or 11, or a nucleotide sequence having at least 80% sequence identity thereto.

[0239] In certain embodiments of any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first molecule may further comprise a first presenting peptide fused to the first B2M polypeptide via a third linker, and / or (b) the second molecule may further comprise a second presenting peptide fused to the second B2M polypeptide via a fourth linker. As used herein, the term "presenting peptide" may refer to a short, e.g., 8-10 amino acid, polypeptide that can non-covalently associate in the protein groove, e.g., a polypeptide formed by a class I HLA molecule in association with B2M.

[0240] In some embodiments, (a) the first presented peptide may be fused to a first B2M polypeptide and (b) the second presented peptide may be fused to a second B2M polypeptide.

[0241] In some embodiments, the first and / or second presentation peptides are the same. In some embodiments, the first and / or second presentation peptides are different.

[0242] In some embodiments, the first and / or second presentation peptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 or 23, or in certain embodiments, the amino acid sequence of SEQ ID NO: 4 or 23. In some embodiments, the first and / or second presentation peptide comprises an amino acid sequence of SEQ ID NO: 4 or 23, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the first and / or second presentation peptide comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5 or 24, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 5 or 24. In some embodiments, the nucleotide sequence encoding the first and / or second presentation peptide comprises a nucleotide sequence of SEQ ID NO: 5 or 24, or a nucleotide sequence having at least 80% sequence identity thereto.

[0243] In various embodiments, the chimeric single chain HLA-E and HLA-G molecules of the present disclosure may include, for example, a first, a second, a third, a fourth, and / or at least one autoprotease peptide linker. In certain embodiments, the linker may be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and Thr. The linker may have a length that is sufficient to link any of the various domains of the chimeric single chain HLA-E and HLA-G molecules such that they form the correct conformation relative to one another to retain a desired activity, such as insertion into a membrane. The linker may be about 5-50 amino acids in length. In some embodiments, the linker is about 10-40 amino acids in length. In some embodiments, the linker is about 10-35 amino acids in length. In some embodiments, the linker is about 10-30 amino acids in length. In some embodiments, the linker is about 10-25 amino acids in length. In some embodiments, the linker is about 10-20 amino acids in length. In some embodiments, the linker is about 15-20 amino acids in length. Exemplary linkers that can be used are Gly-rich linkers, Gly and Ser-containing linkers, Gly and Ala-containing linkers, Ala and Ser-containing linkers, and other flexible linkers. Non-limiting examples of peptide linkers that can be used in accordance with the present disclosure are described in Tables 4 and 6. Additional linkers are described, for example, in International Patent Publication No. WO2019 / 060695, which is incorporated herein by reference in its entirety for all intended purposes.

[0244] [Table 4]

[0245] In some embodiments, the first, second, third, fourth, and / or at least one autoprotease peptide linker each separately comprises an amino acid sequence at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence set forth in Table 4. In some embodiments, the first, second, third, fourth, and / or at least one autoprotease peptide linker each separately comprises an amino acid sequence set forth in Table 4, or an amino acid sequence having at least 80% sequence identity thereto.

[0246] In some embodiments, the first peptide linker and / or the second peptide linker comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6, 39 or 41, or in certain embodiments, the amino acid sequence of SEQ ID NO: 6, 39 or 41. In some embodiments, the first peptide linker and / or the second peptide linker comprises an amino acid sequence of SEQ ID NO: 6, 39 or 41, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the first peptide linker and / or the second peptide linker comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6, or in certain embodiments, the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first peptide linker and / or the second peptide linker comprises an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the first peptide linker and / or the second peptide linker comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7 or 8, or in certain embodiments the nucleotide sequence of SEQ ID NO: 7 or 8. In some embodiments, the nucleotide sequence encoding the first peptide linker and / or the second peptide linker comprises the sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 80% sequence identity thereto.

[0247] In some embodiments, the third peptide linker and / or the fourth peptide linker comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12, or in certain embodiments, the amino acid sequence of SEQ ID NO: 12. In some embodiments, the third peptide linker and / or the fourth peptide linker comprises an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the third peptide linker and / or the fourth peptide linker comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13 or 14, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 13 or 14. In some embodiments, the nucleotide sequence encoding the third peptide linker and / or the fourth peptide linker comprises the sequence of SEQ ID NO: 13 or 14, or a nucleotide sequence having at least 80% sequence identity thereto.

[0248] In some embodiments, at least one of the autoprotease peptide linkers is 5' to the autoprotease peptide, 3' to the autoprotease peptide, or both 5' and 3' to the autoprotease peptide.

[0249] In some embodiments, the autoprotease peptide linker comprises an amino acid sequence of GSG (Gly-Ser-Gly). In some embodiments, the autoprotease peptide linker comprises a GSG linker. In some embodiments, the autoprotease peptide linker comprises any of the various peptide linker sequences disclosed herein, or a combination thereof.

[0250] In certain embodiments of any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein, (a) the first molecule further comprises a first signal peptide operably linked to the HLA-E heavy chain, and / or (b) the second molecule further comprises a second signal peptide operably linked to the HLA-G heavy chain.

[0251] Any of a variety of signal peptides known in the art, such as, for example, any of those described at signalpeptide.com / index.php?m=listspdb_mammalia, which is incorporated by reference for all intended purposes, or functional fragments or combinations thereof, can be used in the practice of the present disclosure.

[0252] In some embodiments, the first signal peptide and the second signal peptide are the same. In some embodiments, the first signal peptide and the second signal peptide are different.

[0253] In some embodiments, the first signal peptide and / or the second signal peptide comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, or in certain embodiments, the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first signal peptide and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the first signal peptide and / or the second signal peptide comprises a nucleotide sequence that is at least 80%, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 3, or in certain embodiments the nucleotide sequence of SEQ ID NO: 2 or 3. In some embodiments, the nucleotide sequence encoding the first signal peptide and / or the second signal peptide comprises the sequence of SEQ ID NO: 2 or 3, or a nucleotide sequence having at least 80% sequence identity thereto.

[0254] In some embodiments, the first signal peptide and the second signal peptide comprise an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, or in certain embodiments, the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first signal peptide and the second signal peptide comprise the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the first signal peptide and the second signal peptide comprises a nucleotide sequence that is at least 80%, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 3, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 2 or 3. In some embodiments, the nucleotide sequence encoding the first signal peptide and the second signal peptide comprises the sequence of SEQ ID NO: 2 or 3, or a nucleotide sequence having at least 80% sequence identity thereto.

[0255] In some embodiments, the presenting peptides and / or signal peptides disclosed herein may be derived from any of the classical HLA I alpha chain (e.g., HLA-A, HLA-B, HLA-C, and HLA-G) signal peptides known in the art. In some embodiments, the presenting peptides and / or signal peptides disclosed herein may be derived from histone H2A.

[0256] In certain embodiments of any of the chimeric single chain HLA-E and HLA-G molecules disclosed herein, the first molecule comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17 or 19, or in certain embodiments, the amino acid sequence of SEQ ID NO: 17 or 19. In some embodiments, the first molecule comprises the amino acid sequence of SEQ ID NO: 17 or 19, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the first molecule comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18 or 20, or in certain embodiments the nucleotide sequence of SEQ ID NO: 18 or 20. In some embodiments, the nucleotide sequence encoding the first molecule comprises the sequence of SEQ ID NO: 18 or 20, or a nucleotide sequence having at least 80% sequence identity thereto.

[0257] In some embodiments, the second molecule comprises an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27 or 29, or in certain embodiments, the amino acid sequence of SEQ ID NO: 27 or 29. In some embodiments, the second molecule comprises an amino acid sequence of SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the second molecule comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28 or 30, or in certain embodiments the nucleotide sequence of SEQ ID NO: 28 or 30. In some embodiments, the nucleotide sequence encoding the second molecule comprises the sequence of SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity thereto.

[0258] In some embodiments, the chimeric single chain HLA-E and HLA-G molecules comprise an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, 165 or 168, or in certain embodiments, the amino acid sequence of SEQ ID NO: 31, 165 or 168. In some embodiments, the chimeric single chain HLA-E and HLA-G molecules comprise an amino acid sequence of SEQ ID NO: 31, 165 or 168, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the chimeric single chain HLA-E and HLA-G molecules comprises a nucleotide sequence that is at least 80%, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32, 120, 166, 167 or 169, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 32, 120, 166, 167 or 169. In some embodiments, the nucleotide sequence encoding the chimeric single chain HLA-E and HLA-G molecules comprises a nucleotide sequence of SEQ ID NO: 32, 120, 166, 167 or 169, or a nucleotide sequence having at least 80% sequence identity thereto.

[0259] In some embodiments, the chimeric single chain HLA-E and HLA-G molecules comprise an amino acid sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, or in certain embodiments, the amino acid sequence of SEQ ID NO: 31. In some embodiments, the chimeric single chain HLA-E and HLA-G molecules comprise an amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 80% sequence identity thereto. In some embodiments, the nucleotide sequence encoding the chimeric single chain HLA-E and HLA-G molecules comprises a nucleotide sequence that is at least 80%, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32, 120 or 167, or in certain embodiments the nucleotide sequence of SEQ ID NO: 32, 120 or 167. In some embodiments, the nucleotide sequence encoding the chimeric single chain HLA-E and HLA-G molecules comprises a nucleotide sequence of SEQ ID NO: 32, 120 or 167, or a nucleotide sequence having at least 80% sequence identity thereto.

[0260] Non-limiting examples of polynucleotides encoding chimeric single chain HLA-E and HLA-G molecules In certain aspects, the present disclosure provides a polynucleotide sequence encoding any of the chimeric single-chain HLA-E and HLA-G molecules disclosed herein. The single-chain HLA-E and HLA-G molecules may, for example, comprise (a) a first molecule comprising an HLA-E heavy chain and (b) a second molecule comprising an HLA-G heavy chain, and (c) a linking peptide between (a) and (b). In some embodiments, the polynucleotide may comprise a nucleotide sequence encoding the first molecule. In some embodiments, the polynucleotide may comprise a nucleotide sequence encoding the second molecule. In some embodiments, the polynucleotide may comprise a nucleotide sequence encoding any of the chimeric single-chain HLA-E and HLA-G molecules disclosed herein.

[0261] In some embodiments, the polynucleotide sequence encoding the first molecule of the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18 or 20, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 18 or 20. In some embodiments, the polynucleotide sequence encoding the first molecule comprises the sequence of SEQ ID NO: 18 or 20, or a nucleotide sequence having at least 80% sequence identity thereto.

[0262] In some embodiments, the polynucleotide sequence encoding the second molecule of the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28 or 30, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 28 or 30. In some embodiments, the polynucleotide sequence encoding the second molecule comprises the sequence of SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity thereto.

[0263] In some embodiments, the polynucleotide sequences encoding the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprise a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32, 120, 166, 167 or 169, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 32, 120, 166, 167 or 169. In some embodiments, the polynucleotide sequences encoding the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 32, 120, 166, 167 or 169, or has at least 80% sequence identity thereto. In some embodiments, the polynucleotide sequences encoding the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprise a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32, 120 or 167, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 32, 120 or 167. In some embodiments, the polynucleotide sequences encoding the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprise a nucleotide sequence of SEQ ID NO: 32, 120 or 167, or a nucleotide sequence having at least 80% sequence identity thereto.

[0264] In some embodiments, a polynucleotide of the present disclosure may be a DNA molecule.

[0265] In some embodiments, a polynucleotide of the present disclosure can be an RNA molecule.

[0266] vector The present disclosure further provides a recombinant vector comprising a polynucleotide encoding a chimeric single-chain HLA-E and HLA-G molecule comprising the polynucleotide encoding the protein disclosed above. In certain embodiments, the polynucleotide is operably linked to at least one regulatory element disclosed herein.

[0267] In some embodiments, a recombinant vector of the present disclosure comprises a polynucleotide sequence encoding a chimeric single chain HLA-E and HLA-G molecule disclosed herein that comprises a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32, 120, 166, 167 or 169, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 32, 120, 166, 167 or 169. In some embodiments, the polynucleotide sequence encoding the chimeric single chain HLA-E and HLA-G molecule comprises a nucleotide sequence that comprises a sequence of SEQ ID NO: 32, 120, 166, 167 or 169, or a nucleotide sequence that has at least 80% sequence identity thereto. In some embodiments, the polynucleotide sequences encoding the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprise a nucleotide sequence that is at least 80%, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32, 120 or 167, or in certain embodiments, the nucleotide sequence of SEQ ID NO: 32, 120 or 167. In some embodiments, the polynucleotide sequences encoding the chimeric single chain HLA-E and HLA-G molecules disclosed herein comprise a nucleotide sequence of SEQ ID NO: 32, 120 or 167, or a nucleotide sequence having at least 80% sequence identity thereto.

[0268] In some embodiments, the polynucleotide sequence encoding the single chain HLA-E and HLA-G molecules may be operably linked to one or more promoters. In some embodiments, the polynucleotide sequence encoding the single chain HLA-E and HLA-G molecules may be operably linked to one, two, three, four, five or more promoters. In some embodiments, the polynucleotide sequence encoding the single chain HLA-E and HLA-G molecules may be operably linked to a single promoter.

[0269] In some embodiments, the one or more promoters are exogenous promoters. In some embodiments, the one or more exogenous promoters can include, for example, CMV, EFla, PGK, CAG, UBC, SV40, human beta actin, or other constitutive, inducible, time-specific, tissue-specific, or cell type-specific promoters. In some embodiments, the one or more promoters are exogenous promoters. In some embodiments, the endogenous promoter can be included in a selected site, for example, AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL, or other loci that meet the criteria of genomic safe harbor.

[0270] In some embodiments, the promoter is an inducible promoter.

[0271] In some embodiments, the promoter is a CAG promoter.In some embodiments, the CAG promoter comprises at least 80% identical polynucleotide sequence to SEQ ID NO:92, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence.

[0272] In some embodiments, the recombinant vector comprising the polynucleotide disclosed herein is a viral vector.By way of non-limiting example, the viral vector can be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphaviral vector, a herpes viral vector, a baculoviral vector, or a vaccinia viral vector.

[0273] In some embodiments, the recombinant vector comprising the polynucleotide disclosed herein is a non-viral vector.As a non-limiting example, the non-viral vector can be minicircle plasmid, Sleeping Beauty transposon, piggyBac transposon, or single-stranded or double-stranded DNA molecule that can be used as template for gene editing based on homology-directed repair (HDR).

[0274] host cell In one aspect, the present disclosure provides an isolated host cell comprising any of the various polynucleotides disclosed herein. In another aspect, the present disclosure provides an isolated host cell comprising any of the various recombinant vectors disclosed herein. In yet another aspect, the present disclosure provides an isolated host cell comprising any of the various HLA-E and HLA-G molecules encoded by the polynucleotides disclosed herein.

[0275] In some embodiments, an isolated host cell disclosed herein may contain two or more polynucleotides or recombinant vectors disclosed herein.

[0276] In some embodiments, the host cell may be an iPSC or a population thereof. In some embodiments, the host cell may be an immune cell.

[0277] In certain aspects, the present disclosure provides immune effector cells or populations thereof derived from the induced pluripotent stem cells (iPSCs) disclosed herein.

[0278] In some embodiments, the isolated host cells, immune effector cells, or populations thereof may be T cells, natural killer (NK) cells, natural killer T cells (NKT cells), mesenchymal stem cells (MSCs), or macrophages.

[0279] In some embodiments, the isolated host cells, immune effector cells, or populations thereof may be T cells. T cells include, but are not limited to, for example, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, for example, T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+ CD8+ T cells, or any other subset of T cells. Other illustrative populations of T cells suitable for use in certain embodiments include naive T cells, memory T cells, and NKT cells.

[0280] In some embodiments, the T cell can be a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an αβ T cell receptor (TCR) T cell, an invariant natural killer T (iNKT) cell, a γδ T cell, a memory T cell, a memory T stem cell (TSCM), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

[0281] In some embodiments, the isolated host cells, immune effector cells, or populations thereof are αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, memory T stem cells (TSCM), naive T cells, effector T cells, T helper cells, or regulatory T cells (Tregs).

[0282] In some embodiments, the host cells, immune effector cells, or populations thereof may be NK cells.

[0283] VII. HLA-E and HLA-G Transgenes According to certain embodiments, the immune cells or iPSCs of the present application can be modified by introducing an exogenous polynucleotide encoding one or more proteins associated with immune escape, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).

[0284] In certain embodiments, iPSCs are engineered by insertion of HLA-E and HLA-G transgenes using the MAD7 / gRNA ribonucleoprotein (RNP) complex described herein. In some embodiments, the HLA-E and HLA-G transgenes disclosed herein can be inserted utilizing the RNP complex, guide sequence and homology arms according to the present disclosure.

[0285] MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and vector systems In certain aspects, the disclosure provides a MAD7 / gRNA ribonucleoprotein (RNP) complex composition for insertion of HLA-E and HLA-G transgenes, the MAD7 / gRNA ribonucleoprotein (RNP) complex composition comprising: (I) MAD7 nuclease; (II) a guide RNA (gRNA) specific for MAD7 nuclease, wherein the gRNA comprises a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, the guide sequence being selected from SEQ ID NOs: 109-119, and wherein the guide sequence is capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell when the gRNA is complexed with the MAD7 nuclease; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to the left and right arms of a target sequence in the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter; (3) a polynucleotide encoding an HLA-E and HLA-G transgene comprising any of the polynucleotides disclosed herein, e.g., a polynucleotide encoding a chimeric single chain HLA-E and HLA-G molecule disclosed herein, to which the promoter is operably linked; and (4) a transcription terminator sequence.

[0286] In certain aspects, the disclosure provides a MAD7 / gRNA ribonucleoprotein (RNP) complex composition for insertion of HLA-E and HLA-G transgenes, the MAD7 / gRNA ribonucleoprotein (RNP) complex composition comprising I) a MAD7 nuclease system, encoded by one or more vectors, the one or more vectors comprising (a) a sequence encoding a guide RNA (gRNA) operably linked to a first regulatory element, the gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, or CLYBL locus in a cell, the guide sequence being selected from SEQ ID NOs: 109-119, and wherein when transcribed, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence. and (II) an HLA-E and HLA-G transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of a target sequence at the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33 or CLYBL locus; (2) a promoter; (3) a polynucleotide encoding an HLA-E and HLA-G transgene comprising any of the polynucleotides disclosed herein, e.g., a polynucleotide encoding a chimeric single chain HLA-E and HLA-G molecule disclosed herein, to which the promoter is operably linked; and (4) a transcription terminator sequence.

[0287] In certain aspects, the disclosure provides a MAD7 / gRNA ribonucleoprotein (RNP)-based vector system, the MAD7 / gRNA ribonucleoprotein (RNP)-based vector system comprising: (I) one or more vectors comprising: (a) a sequence encoding a guide RNA (gRNA), the sequence being operably linked to a first regulatory element, the gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, the gRNA guide sequence being selected from SEQ ID NOs: 109-119, wherein when transcribed, the guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; (b) a sequence encoding a MAD7 nuclease; and (II) an HLA-E and HLA-G transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of a target sequence at the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter; (3) a polynucleotide encoding any of the polynucleotides disclosed herein, e.g., a polynucleotide encoding a chimeric single chain HLA-E and HLA-G molecule disclosed herein, to which the promoter is operably linked; and (4) a transcription terminator sequence.

[0288] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex composition and / or vector system disclosed herein, the cell can be any of the various cells disclosed herein. In some embodiments, the cell can be an induced pluripotent stem cell (iPSC).

[0289] In some embodiments, the first and / or second regulatory element is any of a variety of promoters disclosed herein.

[0290] In some embodiments, the first regulatory element and the second regulatory element are the same. In some embodiments, the first regulatory element and the second regulatory element are different.

[0291] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the gRNA guide sequence is specific for the AAVS1 locus. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 109.

[0292] In some embodiments, the gRNA guide sequence is specific for B2M. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 110.

[0293] In some embodiments, the gRNA guide sequence is specific for the CIITA locus. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 111 or 112.

[0294] In some embodiments, the gRNA guide sequence is specific for the NKG2A locus. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 114.

[0295] In some embodiments, the gRNA guide sequence is specific for the TRAC locus. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 115.

[0296] In some embodiments, the gRNA guide sequence is specific for the CD70 locus. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 116.

[0297] In some embodiments, the gRNA guide sequence is specific for the CD38 locus. In some embodiments, the gRNA guide sequence comprises SEQ ID NO: 117.

[0298] In some embodiments, the gRNA guide sequence is specific for the CD33 locus. In some embodiments, the gRNA guide sequence is specific for the CD33 locus and comprises SEQ ID NO: 118 or 119.

[0299] In some embodiments, the gRNA guide sequence is specific for the CLYBL locus. In some embodiments, the gRNA guide comprises SEQ ID NO: 113.

[0300] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of AAVS1 comprise the nucleotide sequences of SEQ ID NOs: 73 and 74, respectively, or fragments thereof.

[0301] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of B2M comprise the nucleotide sequences of SEQ ID NOs: 76 and 77, respectively, or fragments thereof.

[0302] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CIITA comprise the nucleotide sequences of (i) SEQ ID NOs: 79 and 80, respectively, or (ii) SEQ ID NOs: 95 and 96, respectively, or fragments thereof.

[0303] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of NKG2A comprise the nucleotide sequences of SEQ ID NOs: 85 and 86, respectively, or fragments thereof.

[0304] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of TRAC comprise the nucleotide sequences of SEQ ID NOs: 88 and 89, respectively, or fragments thereof.

[0305] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CD70 comprise the nucleotide sequences of SEQ ID NOs: 98 and 99, respectively, or fragments thereof.

[0306] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, the left and right polynucleotide sequences that are homologous to the left and right arms of the target sequence of CLYBL comprise the nucleotide sequences of SEQ ID NOs: 82 and 83, respectively, or fragments thereof.

[0307] In some embodiments of the MAD7 / gRNA ribonucleoprotein (RNP) complex compositions and / or vector systems disclosed herein, when the RNP complex is introduced into a cell, expression of an endogenous gene that comprises a target sequence complementary to the guide sequence of the gRNA molecule is reduced or eliminated in the cell.

[0308] In some embodiments, expression of an endogenous gene comprising a target sequence complementary to a guide sequence of a gRNA molecule is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, epiregulin activity is reduced by about 5%-20%, 10%-30%, 20%-40%, 30%-50%, 40%-60%, 50%-70%, 60%-80%, 70%-90%, 80%-95% or more. In some embodiments, expression of an endogenous gene comprising a target sequence complementary to a guide sequence of a gRNA molecule is reduced by about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0309] In some embodiments, when the RNP complex is introduced into a cell, expression of an endogenous gene comprising a target sequence complementary to the guide sequence of the gRNA molecule is silenced in the cell.

[0310] In certain aspects, the present disclosure provides one or more retroviruses that constitute the vector system.

[0311] In certain aspects, the present disclosure provides an isolated host cell disclosed herein that can be transformed by the vector disclosed herein.In certain aspects, the present disclosure provides an isolated host cell disclosed herein that is transformed by one or more retroviruses disclosed herein.In some embodiments, the host cell can be any of the various host cells disclosed herein.

[0312] In some embodiments, the host cell may be an iPSC or a population thereof. In some embodiments, the host cell may be an immune cell.

[0313] In certain aspects, the present disclosure provides immune effector cells or populations thereof derived from the induced pluripotent stem cells (iPSCs) disclosed herein.

[0314] In some embodiments, the isolated host cells, immune effector cells, or populations thereof may be T cells, natural killer (NK) cells, natural killer T cells (NKT cells), mesenchymal stem cells (MSCs), or macrophages.

[0315] In some embodiments, the isolated host cells, immune effector cells, or populations thereof may be T cells. T cells include, but are not limited to, for example, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, for example, T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+ CD8+ T cells, or any other subset of T cells. Other illustrative populations of T cells suitable for use in certain embodiments include naive T cells, memory T cells, and NKT cells.

[0316] In some embodiments, the T cell can be a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an αβ T cell receptor (TCR) T cell, an invariant natural killer T (iNKT) cell, a γδ T cell, a memory T cell, a memory T stem cell (TSCM), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

[0317] In some embodiments, the isolated host cells, immune effector cells, or populations thereof are αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, memory T stem cells (TSCM), naive T cells, effector T cells, T helper cells, or regulatory T cells (Tregs).

[0318] In some embodiments, the host cells, immune effector cells, or populations thereof may be NK cells.

[0319] In some embodiments, the isolated host cells, immune effector cells, or populations thereof disclosed herein exert improved protective effects against allogeneic cell lysis compared to cells lacking the chimeric single chain HLA-E and HLA-G molecules of the present disclosure or the polynucleotides of the present disclosure. The cell lysis can be mediated by allogeneic effector cells present in peripheral blood mononuclear cells (PBMCs). The effect is primarily caused by NK cells, although other immune cells may also contribute.

[0320] VIII. Chimeric Antigen Receptor ("CAR") Transgenes In some embodiments, at least one of the transgenes that may be inserted into a particular locus of the immune cells or iPSCs of the present application encodes an exogenous chimeric antigen receptor (CAR), e.g., a CAR that targets a tumor antigen.

[0321] As used herein, a "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide that includes at least an extracellular domain that specifically binds to a target (e.g., an antigen), a transmembrane domain, and an intracellular signaling domain. The association of the extracellular domain of the CAR with a target antigen on the surface of a target cell results in clustering of the CAR, providing an activation stimulus to the CAR-containing cell. The CAR redirects the specificity of immune effector cells in a major histocompatibility complex (MHC)-independent manner, inducing the production of molecules that can mediate proliferation, cytokine production, phagocytosis, and / or cell death of target antigen-expressing cells.

[0322] As used herein, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of a nascent polypeptide, such as the HLA-E and HLA-G polypeptides or chimeric antigen receptor (CAR) polypeptides disclosed herein, that can direct the nascent protein to the endoplasmic reticulum and subsequently direct surface expression or secretion.

[0323] As used herein, the term "extracellular domain," "extracellular antigen-binding domain," or "extracellular ligand-binding domain" refers to the portion of a CAR that is located on the outside of the cell membrane and can bind to a target, such as an antigen or ligand.

[0324] As used herein, the term "hinge region" or "hinge domain" refers to a portion of a CAR that connects the two adjacent domains of the CAR, i.e., the extracellular domain and the transmembrane domain of the CAR.

[0325] As used herein, the term "transmembrane domain" refers to the portion of a transmembrane molecule (e.g., a CAR or HLA-E and HLA-G molecule of the present application) that spans across the cell membrane and anchors the molecule to the cell membrane.

[0326] As used herein, the terms "intracellular signaling domain," "cytoplasmic signaling domain" or "intracellular signaling domain" refer to the portion of a CAR that is located inside the cell membrane and is capable of transmitting an effector signal.

[0327] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., a T cell) that provides a primary cytoplasmic signaling sequence that regulates primary activation of a receptor in a manner that stimulates at least some aspect of an immune cell signaling pathway. Stimulatory molecules contain two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation (termed "primary signaling domains") and those that act antigen-independently to provide a costimulatory secondary signal (termed "costimulatory signaling domains").

[0328] In certain embodiments, the extracellular domain comprises an antigen-binding domain. The antigen-binding domain can be, for example, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen. The antigen-binding domain of the present application possesses one or more desirable functional properties, including, but not limited to, high affinity binding to tumor antigen; high specificity for tumor antigen; ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing tumor antigen; and ability to inhibit tumor growth when administered alone or in combination with other anti-cancer therapies in subjects and animal models in need thereof.

[0329] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including human, humanized, composite and chimeric antibodies and antibody fragments, which are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a particular antigen. Antibody structure is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM) depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Thus, the subject antibodies can be of any of the five major classes or corresponding subclasses. Preferably, the subject antibodies are IgG1, IgG2, IgG3 or IgG4. The antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Thus, the antibodies of the present application may contain kappa or lambda light chain constant domains. According to certain embodiments, the antibodies of the present application contain heavy and / or light chain constant regions from a rat or human antibody. In addition to the heavy and light chain constant domains, the antibodies contain an antigen binding region made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2 and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2 and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2 and HCDR3.

[0330] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a particular tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0331] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the present application may be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or by recombinant DNA methods. For example, monoclonal antibodies may be produced by hybridomas that contain B cells obtained from transgenic non-human animals, such as transgenic mice or rats, whose genomes contain human heavy chain transgenes and light chain transgenes.

[0332] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), single domain antibody (sdAb), scFv dimer (bivalent diabody); multispecific antibody formed from a portion of an antibody containing one or more CDRs; camelized single domain antibody, minibody, nanobody, domain antibody, bivalent domain antibody, light chain variable domain (VL); camelid antibody variable domain (VHH); or any other antibody fragment that binds to an antigen that does not include a complete antibody structure. An antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment binds.

[0333] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).

[0334] As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the art, comprising a heavy chain variable region and a heavy chain constant region, or comprising only a heavy chain variable region.

[0335] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0336] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology to the sequence of a human antibody, thereby retaining the antigen-binding properties of the antibody but reducing its antigenicity in the human body.

[0337] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity and capacity, while the constant regions correspond to the sequences of antibodies derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0338] As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or substantially do not overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In an embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In certain embodiments, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0339] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or no more than two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises an scFv or fragment thereof having binding specificity for a first epitope and an scFv or fragment thereof having binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a VFv or fragment thereof having binding specificity for a first epitope. H H and V with binding specificity for a second epitope. H Contains H.

[0340] As used herein, an antigen-binding domain that "specifically binds to a tumor antigen" is one that binds to a tumor antigen at least 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×10 -10KD refers to an antigen-binding domain that binds with a KD equal to or less than M. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using methods in the art in light of the present disclosure. For example, the KD of an antigen-binding domain can be determined by using surface plasmon resonance, for example, by using a biosensor system, such as a Biacore® system, or by using biolayer interference technology, such as the Octet RED96 system.

[0341] The smaller the KD value of an antigen-binding domain, the higher the affinity with which the antigen-binding domain binds to a target antigen.

[0342] In various embodiments, antibodies or antibody fragments suitable for use in the CARs of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single chain Fvs (scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals ("SMIPs™"), intrabodies, minibodies, single domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies against an antigen-specific TCR), and epitope-binding fragments of any of the above. The antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0343] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single domain antibody (sdAb) or nanobody, a dAb fragment, an Fd' fragment, an Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is a scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0344] In some embodiments, the CAR is part of a universal chimeric antigen receptor system (arCAR) with adaptable receptor specificity components. The arCAR system may include: (i) an immune effector cell having a chimeric antigen receptor comprising a first polypeptide comprising (a) an extracellular tag-binding domain, (b) a transmembrane domain, and (c) at least one intracellular signaling domain; and (ii) a second polypeptide comprising (a) an antigen-binding domain that binds to at least one antigen on a target cell and (b) a tag recognized by the extracellular tag-binding domain, where (i) the tag comprises an antibody, an antigen-binding fragment thereof, or an alternative scaffold, and the extracellular domain comprises an anti-idiotypic molecule that binds to the tag, or (ii) the tag comprises an anti-idiotypic molecule that binds to the extracellular tag-binding domain, and the extracellular tag-binding domain comprises an antibody, an antigen-binding fragment thereof, or an alternative scaffold.

[0345] In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises a single domain antibody or a nanobody.

[0346] In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.

[0347] In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH.

[0348] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH.

[0349] In some embodiments, at least one of the extracellular tag binding domain, the antigen binding domain, or the tag comprises an scFv.

[0350] In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.

[0351] In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise an scFv.

[0352] Alternative scaffolds of immunoglobulin domains that exhibit similar functional characteristics, such as high affinity and specific binding to target biomolecules, can also be used in the CARs of the present disclosure. Such scaffolds have been shown to produce molecules with improved characteristics, such as greater stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that can be used in the CARs of the disclosure include engineered tenascin-derived, tenascin type III domains (e.g., Centyrin™); engineered gamma-B crystallin-derived scaffolds or engineered ubiquitin-derived scaffolds (e.g., Affilin); engineered fibronectin-derived, 10th fibronectin type III (10Fn3) domains (e.g., monobodies, AdNectins™, or AdNexins™); engineered ankyrin repeat motif-containing polypeptides (e.g., DARPins™); engineered low density lipoprotein receptor-derived, A domain (LDLR-A) (e.g., Avimers™); lipocalins (e.g., anticalins); engineered protease inhibitors (e.g., ribozymes ... Kunitz domains from protease inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2); engineered Z domains from Protein A (Affibodies™); Sac7d-derived polypeptides (e.g., Nanoffitins® or Affitins); engineered SH2 domains from Fyn (e.g., Fynomers®); CTLD3 (e.g., Tetranectin); Thioredoxins (e.g., peptide aptamers); KALBITOR®; β-sandwiches (e.g., iMabs); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimetics; and genetically engineered counterparts of any of the foregoing that retain their binding functionality (Worn A, Pluckthun A, J Mol Biol 305: 989-1010 (2001);Xu L et al., Chem Biol 9: 933-42 (2002);Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004);Binz H et al., Nat Biolechnol 23: 1257-68 (2005);Hey T et al., Trends Biotechnol 23:514-522 (2005);Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005);Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006);Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007);Skerra, Current Opin. in Biotech., 2007 18: 295-304;Byla P et al., J Biol Chem 285: 12096 (2010);Zoller F et al., Molecules 16: 2467-85 (2011), each of which is incorporated herein by reference in its entirety for all intended purposes.

[0353] In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0354] In some embodiments, the CAR of the present disclosure comprises a leader sequence. The leader sequence may be located at the N-terminus of the extracellular antigen binding domain. The leader sequence may be optionally cleaved from the extracellular antigen binding domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of leader sequences known to those skilled in the art may be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte macrophage colony stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of T cells. In certain embodiments, the leader sequence is derived from human immunoglobulin heavy chain (HC).

[0355] In some embodiments, the leader sequence is derived from GMCSFR. In one embodiment, the GMCSFR leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 133, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 133.

[0356] In some embodiments, a CAR of the present disclosure comprises a transmembrane domain fused in-frame between the extracellular domain and the cytoplasmic domain.

[0357] The transmembrane domain may be derived from a protein that contributes to the extracellular domain, a protein that contributes to the signal transduction or co-signal transduction domain, or may be obtained by a completely different protein. In some cases, the transmembrane domain may be selected or modified by amino acid substitution, deletion or insertion to minimize interaction with other members of the CAR complex. In some cases, the transmembrane domain may be selected or modified by amino acid substitution, deletion or insertion to avoid binding of the protein that naturally accompanies the transmembrane domain. In certain embodiments, the transmembrane domain comprises additional amino acids to allow flexibility and / or optimal distance between the domains connected to the transmembrane domain.

[0358] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains that are particularly useful in the present disclosure may be derived from (i.e., may include at least) the α, β or ζ chain of the T cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137 or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will mainly comprise hydrophobic residues, such as leucine and valine. For example, triplets of phenylalanine, tryptophan and / or valine may be found at each end of a synthetic transmembrane domain.

[0359] In some embodiments, it may be desirable to utilize transmembrane domains of the zeta, eta, or FcεR1 gamma chains that contain cysteine ​​residues capable of disulfide bonding, so that the resulting chimeric protein can form disulfide-linked dimers with itself or with unmodified versions of the zeta, eta, or FcεR1 gamma chains or related proteins. In some cases, the transmembrane domains will be selected or modified by amino acid substitutions to avoid binding of the domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it may be desirable to utilize transmembrane domains of zeta, eta, or FcεR1 gamma and -β, MB1 (Igα.), B29, or CD3-γ, zeta, or eta, to retain physical association with other members of the receptor complex.

[0360] In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 154, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 154. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 155, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 155.

[0361] In some embodiments, a CAR of the present disclosure comprises a hinge region between the extracellular antigen-binding domain and the transmembrane domain, wherein the antigen-binding domain, the hinge region, and the transmembrane domain are in-frame with each other.

[0362] The hinge region may comprise any oligo- or polypeptide that functions to link the extracellular antigen-binding domain to the transmembrane domain. The hinge region may be used to obtain greater flexibility of and accessibility to the antigen-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. The hinge region may be derived from all or a portion of a naturally occurring molecule, for example, from all or a portion of the extracellular region of CD8, CD4 or CD28, or from all or a portion of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge region sequence, or may be a completely synthetic hinge region sequence. Non-limiting examples of hinge regions that can be used in accordance with the present disclosure include portions of the human CD8 alpha chain, the partial extracellular domain of CD28, the FcyRIIIa receptor, IgG, IgM, IgA, IgD, IgE, and Ig hinges, or functional fragments thereof. In some embodiments, additional linking amino acids are added to the hinge region to ensure that the antigen binding domain is at an optimal distance from the transmembrane domain. In some embodiments, if the spacer is derived from an Ig, the spacer can be mutated to prevent Fc receptor binding.

[0363] The hinge domain can be derived from CD8α, CD28, or immunoglobulin (IgG). For example, the IgG hinge can be from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof.

[0364] In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or a functional fragment thereof. In certain embodiments, the IgG hinge is from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof. In certain embodiments, the hinge domain comprises the CH1, CH2, CH3 and / or hinge region of an immunoglobulin. In certain embodiments, the hinge domain comprises the core hinge region of an immunoglobulin. The term "core hinge" may be used synonymously with the term "short hinge" (also known as "SH"). A non-limiting example of a suitable hinge domain is the core immunoglobulin hinge region, including EPKSCDKTHTCPPCP (SEQ ID NO: 68) from IgG1, ERKCCVECPPCP (SEQ ID NO: 69) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 70) from IgG3, and ESKYGPPCPSCP (SEQ ID NO: 71) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes). In certain embodiments, the hinge domain is a fragment of an immunoglobulin hinge.

[0365] In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 152, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 152. In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 153, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 153.

[0366] In some embodiments, the transmembrane domain and / or hinge domain are derived from CD8 or CD28. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and the hinge domain are derived from CD28. Non-limiting exemplary hinge sequences are provided in Table 5.

[0367] [Table 5]

[0368] In certain aspects, the CAR of the present disclosure comprises a cytoplasmic domain that comprises at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more costimulatory signaling domains.

[0369] The cytoplasmic domain is involved in the activation of at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Although the entire signaling domain is usually present, it is often not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, provided that it transmits an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the signaling domain sufficient to transmit an effector function signal.

[0370] Non-limiting examples of signaling domains that can be used in the CARs of the present disclosure include, for example, signaling domains from DAP10, DAP12, Fc epsilon receptor I gamma chain (FCER1G), FcR beta, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

[0371] In some embodiments, the cytoplasmic domain comprises a CD3 zeta signaling domain. In one embodiment, the CD3 zeta signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 137, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 137.

[0372] In some embodiments, the cytoplasmic domain further comprises one or more costimulatory signaling domains, in some embodiments, the one or more costimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.

[0373] In one embodiment, the costimulatory signaling domain is derived from 41BB. In one embodiment, the 41BB costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 139, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 139.

[0374] In one embodiment, the costimulatory signaling domain is derived from IL2Rb. In one embodiment, the IL2Rb costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 140, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 140.

[0375] In one embodiment, the costimulatory signaling domain is derived from CD40. In one embodiment, the CD40 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 141, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 141.

[0376] In one embodiment, the costimulatory signaling domain is derived from OX40. In one embodiment, the OX40 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 142, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 142.

[0377] In one embodiment, the costimulatory signaling domain is derived from CD80. In one embodiment, the CD80 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 143, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 143.

[0378] In one embodiment, the costimulatory signaling domain is derived from CD86. In one embodiment, the CD86 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 144, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 144.

[0379] In one embodiment, the costimulatory signaling domain is derived from CD27. In one embodiment, the CD27 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 145, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 145.

[0380] In one embodiment, the costimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 146, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 146.

[0381] In one embodiment, the costimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 147, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 147.

[0382] In one embodiment, the costimulatory signaling domain is derived from DAP10. In one embodiment, the DAP10 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 148, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 148.

[0383] In one embodiment, the costimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP12 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 149, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 149.

[0384] In one embodiment, the costimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 150, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 150.

[0385] In one embodiment, the costimulatory signaling domain is derived from CD28. In one embodiment, the CD28 costimulatory signaling domain comprises an amino acid sequence set forth in SEQ ID NO: 151, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 151.

[0386] In one embodiment, a CAR of the present disclosure comprises a hinge region, a transmembrane domain, and a costimulatory signaling domain, all derived from CD28. In one embodiment, the hinge region, the transmembrane domain, and the costimulatory signaling domain derived from CD28 comprise the amino acid sequence set forth in SEQ ID NO: 136, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 136.

[0387] In some embodiments, the CAR of the present disclosure comprises one costimulatory signaling domain. In some embodiments, the CAR of the present disclosure comprises two or more costimulatory signaling domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.

[0388] In some embodiments, the signal transduction domain and the costimulatory signal transduction domain can be arranged in any order. In some embodiments, the signal transduction domain is upstream of the costimulatory signal transduction domain. In some embodiments, the signal transduction domain is downstream from the costimulatory signal transduction domain. If more than one costimulatory domain is included, the order of the costimulatory signal transduction domains could be interchanged.

[0389] Non-limiting exemplary CAR regions and sequences are provided in Table 6.

[0390] [Table 6-1]

[0391] [Table 6-2]

[0392] [Table 6-3]

[0393] In some embodiments, the antigen binding domain of the CAR described herein binds to an antigen. The antigen binding domain of the CAR described herein can bind to more than one antigen or more than one epitope in an antigen. For example, the antigen binding domain of the CAR described herein can bind to two, three, four, five, six, seven, eight or more antigens. As another example, the antigen binding domain of the CAR described herein can bind to two, three, four, five, six, seven, eight or more epitopes in the same antigen.

[0394] The selection of antigen binding domain may depend on the type and number of antigens that define the surface of target cells. For example, the antigen binding domain can be selected to recognize an antigen that serves as a cell surface marker on target cells that is associated with a particular disease state. In certain embodiments, the CAR of the present disclosure can be genetically modified to target a tumor antigen of interest by engineering a desired antigen binding domain that specifically binds to the antigen (e.g., on tumor cells). Non-limiting examples of cell surface markers that can serve as targets for the antigen binding domain in the CAR of the present disclosure include those associated with tumor cells or autoimmune diseases.

[0395] In some embodiments, the antigen binding domain binds to at least one tumor antigen or autoimmune antigen.

[0396] In some embodiments, the antigen binding domain binds to at least one tumor antigen. In some embodiments, the antigen binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors.

[0397] In some embodiments, the antigen binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.

[0398] In some embodiments, the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematological malignancies.Non-limiting examples of tumor antigens associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1, and IL13Rα2.Non-limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, mesothelin, CA125, EpCAM, EGFR, PDGFRα, nectin-4, and B7H4.Non-limiting examples of tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigens associated with liver cancer include claudin 18.2, GPC-3, EpCAM, cMET, and AFP.Non-limiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70.Non-limiting examples of tumor antigens associated with bladder cancer include nectin-4 and SLITRK6.

[0399] Additional examples of antigens that may be targeted by the antigen binding domain include alpha-fetoprotein, A3, antigen specific for the A33 antibody, Ba 733, BrE3 antigen, carbonic anhydrase EX, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon specific antigen-p (CSAp), CEA (CEACAM5), CEACAM 6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, E phA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and These include, but are not limited to, its subunits, hypoxia inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-I), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placenta growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A antigen, angiogenesis markers, oncogene markers or oncogene products.

[0400] In one embodiment, the antigen targeted by the antigen binding domain is CD19. In one embodiment, the antigen binding domain comprises an anti-CD19 scFv. In one embodiment, the anti-CD19 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 134, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 134. In one embodiment, the anti-CD19 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 135, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 135. In one embodiment, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 138, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99% sequence identity to SEQ ID NO: 138.

[0401] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells that produce antibodies against "self". In some embodiments, the antigen is associated with an autoimmune disease or disorder, such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease, or ulcerative colitis.

[0402] In some embodiments, autoimmune antigens that can be targeted by the CARs disclosed herein include platelet antigens, myelin protein antigens, Sm antigens in snRNPs, islet cell antigens, rheumatoid factor, and anti-citrullinated proteins, citrullinated proteins and peptides, such as CCP-1, CCP-2 (periodic citrullinated peptides), fibrinogen, fibrin, vimentin, filaggrin, type I and type II collagen peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factors, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage, such as type II, IX and XI collagen, circulating serum proteins, such as RF (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components, such as RA33 / hnRNP. A2, Sm, eukaryotic translation elongation factor 1 alpha 1, stress proteins such as HSP-65, -70, -90, BiP, inflammatory / immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein, Granular proteins such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factor, histones, ribosomal P protein, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonucleopartides and proteins such as Sm antigens (including but not limited to SmD' and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro(SSA) and La(SSB) antigens.

[0403] In various embodiments, the scFv fragments used in the CAR of the present disclosure may include a linker between the VH and VL domains. The linker may be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should have a length that is sufficient to link the VH and VL such that they form the correct conformation relative to each other to retain the desired activity, such as binding to an antigen. The linker may be about 5-50 amino acids in length. In some embodiments, the linker is about 10-40 amino acids in length. In some embodiments, the linker is about 10-35 amino acids in length. In some embodiments, the linker is about 10-30 amino acids in length. In some embodiments, the linker is about 10-25 amino acids in length. In some embodiments, the linker is about 10-20 amino acids in length. In some embodiments, the linker is about 15-20 amino acids in length. Exemplary linkers that can be used are Gly-rich linkers, Gly- and Ser-containing linkers, Gly- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0404] In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises an amino acid sequence as set forth in SEQ ID NO: 33, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 33. In another embodiment, the linker is a (G4S)3 linker (SEQ ID NO: 6). In one embodiment, the (G4S)3 linker (SEQ ID NO: 6) comprises an amino acid sequence as set forth in SEQ ID NO: 6, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 6. In another embodiment, the linker is a (G4S)2 linker (SEQ ID NO: 41). In one embodiment, the (G4S)2 linker (SEQ ID NO: 41) comprises the amino acid sequence set forth in SEQ ID NO: 41, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 41. In another embodiment, the linker is a (G4S)4 linker (SEQ ID NO: 39). In one embodiment, the (G4S)4 linker (SEQ ID NO: 39) comprises the amino acid sequence set forth in SEQ ID NO: 39, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 39.

[0405] Other linker sequences may include portions of the immunoglobulin hinge area, CL or CH1, from any immunoglobulin heavy or light chain isotype. Exemplary linkers that can be used include any of the linkers of the sequences shown in Table 4 or Table 6 of the present disclosure. Additional linkers are described, for example, in International Patent Publication No. WO 2019 / 060695, which is incorporated herein by reference in its entirety for all intended purposes.

[0406] Regulatory elements In certain embodiments, the polynucleotide encoding MAD7 nuclease, gRNA, or exogenous polynucleotide for insertion, such as HLA-E and HLA-G transgene, is operably linked to at least a regulatory element. The regulatory element may be capable of mediating the expression of MAD7, gRNA, and / or transgene in host cell. Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyadenylation (polyA) tails, IRES elements, response elements, and termination elements.

[0407] In some embodiments, the exogenous polynucleotide for insertion is operably linked to: (1) one or more exogenous promoters, including CMV, EFla, PGK, CAG, UBC, SV40, human beta actin, or other constitutive, inducible, time-specific, tissue-specific, or cell type-specific promoters; or (2) one or more endogenous promoters contained at the selected site, such as AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL, or other loci that meet the criteria of the Genomic Safe Harbor.

[0408] In some embodiments, the promoter is a CAG promoter.In some embodiments, the CAG promoter comprises at least 80% identical polynucleotide sequence to SEQ ID NO:92, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence.

[0409] In some embodiments, the exogenous polynucleotide for insertion is operably under the control of a Kozak consensus sequence. In some embodiments, the Kozak sequence comprises a polynucleotide sequence of GCCACC, or a variant thereof.

[0410] In certain embodiments, the exogenous polynucleotide for insertion is operably linked to a terminator / polyadenylation signal.In some embodiments, the terminator / polyadenylation signal is an SV40 signal.In certain embodiments, the SV40 signal comprises at least 80% identical polynucleotide sequence with SEQ ID NO:93, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotide sequence.Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH and PGK.

[0411] Other genome editing needs In other embodiments of the above cells, genome editing utilizing the RNP complexes of the present disclosure may include the insertion of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptide proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, potential drug targets, or proteins that promote the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genomically engineered iPSCs or their derived cells. Other transgene insertions may include those encoding PET receptors, homeostatic cytokines, and inhibitory checkpoint inhibitory proteins, such as PD1, PD-L1, and CTLA4, and proteins that target the CD47 / signal regulatory protein alpha (SIRPα) axis.

[0412] VIII. Composition In another general aspect, the present application provides a composition comprising an isolated polynucleotide of the present application, a host cell of the present application, and / or an iPSC or a derived cell thereof.

[0413] In certain embodiments, the composition further comprises one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector comprising one or more polynucleotides of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (ImiD).

[0414] In certain embodiments, the composition is a pharmaceutical composition comprising the isolated polynucleotide of the present application, the host cell of the present application and / or the iPSC or derived cell thereof and a pharma- ceutically acceptable carrier. The term "pharmaceutical composition" as used herein refers to a product comprising the isolated polynucleotide of the present application, the isolated polypeptide of the present application, the host cell of the present application, and / or the iPSC or derived cell thereof together with a pharma- ceutically acceptable carrier. The polynucleotides, polypeptides, host cells, and / or iPSC or derived cell thereof of the present application and compositions comprising them are also useful for the manufacture of drugs for the therapeutic applications described herein.

[0415] In certain aspects, the invention provides pharmaceutical compositions comprising the isolated host cells disclosed herein. In certain aspects, the invention provides pharmaceutical compositions comprising immune effector cells derived from iPSCs disclosed herein.

[0416] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the compositions described herein or the biological activity of the compositions described herein. According to certain embodiments, in light of the present disclosure, any pharmaceutical acceptable carrier suitable for use in polynucleotides, polypeptides, host cells, and / or iPSCs or derived cells thereof can be used.

[0417] The formulation of pharma- ceutical active ingredients with pharma- ceutical acceptable carriers is described in the art, e.g., in Remington: The Science and Practice of Pharmacy (e.g., 21 stedition (2005), and any subsequent editions). Non-limiting examples of additional components include buffers, diluents, solvents, isotonicity adjusters, preservatives, stabilizers, and chelating agents. One or more pharma- ceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the present application.

[0418] IX.How to use In another general aspect, the present application provides a method for preventing or treating a disease or condition in a subject in need thereof. The method comprises administering to a subject in need thereof a therapeutically effective amount of any of the cells of the present application, e.g., host cells and / or immune effector cells or populations thereof, and / or compositions of the present application, e.g., pharmaceutical compositions. In some embodiments, the composition may be, for example, a pharmaceutical composition comprising an isolated host cell disclosed herein, or an immune effector cell derived from an iPSC.

[0419] In certain embodiments, the disease or condition is cancer.

[0420] In certain aspects, the present disclosure provides a method for preventing or treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of a host cell, immune effector or population thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0421] The cancer can be, for example, solid or liquid cancer. The cancer can be, for example, selected from the group consisting of lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, endometrial cancer, prostate cancer, thyroid cancer, glioma, glioblastoma, and other solid tumors, and non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma / disease (HD), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0422] In some embodiments, the cancer may be, for example, but not limited to, lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer.

[0423] In some embodiments, the cancer is selected from the group consisting of leukemia, e.g., AML, CML, ALL, and CLL, lymphoma, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, and solid cancers, e.g., sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and brain cancer, and cancer of unknown etiology (CUP).

[0424] Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of "cancer cells" as used herein includes not only primary cancer cells, but also any cells derived from cancer progenitor cells. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that usually appear as solid tumors, a "clinically detectable" tumor is one that is detectable based on tumor mass, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during physical examination, and / or is detectable due to the expression of one or more cancer-specific antigens in samples that can be taken from patients.

[0425] Cancerous conditions may be characterized by the abnormal proliferation of malignant cancer cells and may include leukemias, e.g., AML, CML, ALL, and CLL, lymphomas, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, and solid cancers, e.g., sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and brain cancer, and cancer of unknown etiology (CUP).

[0426] Cancer cells in an individual may be immunologically distinct from normal somatic cells in that individual (i.e., cancerous tumors are immunogenic). For example, cancer cells may be capable of eliciting a systemic immune response in an individual against one or more antigens expressed by the cancer cells. The tumor antigen that elicits the immune response may be specific to the cancer cells or may be shared by one or more normal cells in the individual.

[0427] An individual's cancer cells suitable for treatment as described herein may express the antigen and / or may be of the correct HLA type to bind to the antigen receptor expressed by T cells.

[0428] The individual suitable for the above treatment can be a mammal.In a preferred embodiment, the individual is a human.In other preferred embodiments, non-human mammals can be utilized, particularly mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or lagomorph).

[0429] In some embodiments, an individual may have minimal residual disease (MRD) after a first cancer treatment. In some embodiments, an individual may not have minimal residual disease after one or more cancer treatments or repeat administrations.

[0430] An individual with cancer may present with at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks, e.g., Harrison's Principles of Internal Medicine, 15 th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some cases, diagnosing cancer in an individual may involve identifying a particular cell type (e.g., cancer cells) in a sample of bodily fluid or tissue taken from the individual.

[0431] Antitumor effect is a biological effect that can be manifested by a decrease in tumor growth rate, a decrease in tumor volume, a decrease in tumor cell number, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. "Antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies described herein, and of the T cells obtainable according to the method of the present invention, in preventing the development of tumors in the first place.

[0432] Treatment can be any treatment and / or cure, whether of a human or an animal (e.g., in veterinary applications), which achieves some desired therapeutic effect, e.g., inhibiting or slowing the progression of a condition, including slowing the rate of progression, halting the rate of progression, improving the condition, curing or ameliorating the condition (whether partial or complete), preventing, delaying, alleviating or arresting one or more symptoms and / or signs of the condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0433] Treatment can also be preventative (i.e., prophylactic). For example, an individual who is susceptible to or at risk of developing or recurring cancer can be treated as described herein. Such treatment can prevent or delay the development or recurrence of cancer in an individual.

[0434] In particular, the treatment may include inhibiting cancer growth, including complete cancer remission, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indicators that indicate changes in cancer to a more developed form. Thus, indicators for measuring inhibition of cancer growth include reduced cancer cell survival, reduced tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, disruption of tumor vasculature, improved performance in delayed-type skin hypersensitivity tests, increased activity of T cells, and reduced tumor-specific antigen levels. Administration of modified T cells as described herein can improve an individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the individual's propensity for cancer growth.

[0435] According to an embodiment of the present application, the composition comprises a therapeutically effective amount of isolated polynucleotide, isolated polypeptide, host cell, and / or iPSC or its derived cell.As used herein, the term "therapeutically effective amount" refers to the amount of active ingredient or component that induces a desired biological or pharmaceutical response in a subject.The therapeutically effective amount can be determined empirically and in a routine manner in relation to the stated purpose.

[0436] As used herein with respect to the cells and / or pharmaceutical compositions of the present application, a therapeutically effective amount refers to the amount of the cells and / or pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0437] According to certain embodiments, a therapeutically effective amount refers to an amount of treatment that is sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder or condition to be treated or symptoms associated therewith; (ii) shortening the duration of the disease, disorder or condition to be treated or symptoms associated therewith; (iii) preventing the progression of the disease, disorder or condition to be treated or symptoms associated therewith; (iv) causing regression of the disease, disorder or condition to be treated or symptoms associated therewith; (v) preventing the onset or manifestation of the disease, disorder or condition to be treated or symptoms associated therewith; (vi) preventing the onset or manifestation of the disease, disorder or condition to be treated or symptoms associated therewith; ) prevent the recurrence of the disease, disorder or condition to be treated or symptoms associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated or symptoms associated therewith; (viii) shorten the length of hospitalization of a subject having the disease, disorder or condition to be treated or symptoms associated therewith; (ix) extend the duration of survival of a subject having the disease, disorder or condition to be treated or symptoms associated therewith; (xi) inhibit or alleviate the disease, disorder or condition to be treated or symptoms associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic efficacy of another treatment.

[0438] The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, for example, weight, health), whether the subject is a human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0439] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous or intramuscular administration.

[0440] The cells of the present application and / or the pharmaceutical compositions of the present application can be administered in any conventional manner known to those skilled in the art. For example, the cells of the present application can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and / or transplantation. The compositions comprising the cells of the present application can be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intrapleurally, by intravenous (iv) injection, or intraperitoneally. In certain embodiments, the cells of the present application can be administered with or without lymphodepleting the subject.

[0441] Pharmaceutical compositions comprising the cells of the present application may be provided as sterile liquid preparations, usually isotonic aqueous solutions in which the cells are suspended, or as emulsions or dispersions, typically buffered to a selected pH, as needed. The compositions may include carriers suitable for cell integrity and viability and for administration of the cell composition, such as water, saline, and phosphate buffered saline.

[0442] Sterile injectable solutions can be prepared by incorporating the cells of the present application in a suitable amount of a suitable solvent with various other ingredients as desired. Such compositions may contain pharma- ceutically acceptable carriers, diluents or excipients, such as sterile water, saline, glucose or dextrose, suitable for use with cell compositions and for administration to subjects such as humans. Suitable buffers for providing cell compositions are well known in the art. Any vehicle, diluent or additive used is compatible with maintaining the integrity and viability of the cells of the present application.

[0443] The cells and / or pharmaceutical compositions of the present application can be administered in a physiologically acceptable vehicle. The cell population comprising the cells of the present application can include a purified population of cells. Those skilled in the art can easily determine the cells in the cell population using a variety of well-known methods. The range of purity in the cell population comprising the genetically modified cells of the present application can be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art, and for example, an increase in dosage may be required due to a decrease in purity.

[0444] The cells of the present application are generally administered as a dose based on the number of cells per kilogram (cells / kg) of body weight of the subject to whom the cells are administered and / or the subject to whom a pharmaceutical composition containing the cells is administered. Generally, cell doses range from about 10 to about 10, depending on the method and location of administration. 4 ~about 10 10 Cells / kg body weight, e.g., about 10 5 ~about 10 9 , about 10 5 ~about 10 8 , about 10 5 ~about 10 7 , or about 10 5 ~about 10 6 Generally, higher doses are used for systemic administration than for local administration, where the immune cells of the present application are administered to the area of ​​the tumor and / or cancer. Exemplary dose ranges include 1×10 4 ~1×10 8 , 2×10 4 ~1×10 8 , 3×10 4 ~1×10 8 , 4×10 4 ~1×10 8 , 5×10 4 ~6×10 8 , 7×10 4 ~1×10 8 , 8×10 4 ~1×10 8 , 9×10 4 ~1×108 、1×10 5 ~1×10 8 、1×10 5 ~9×10 7 、1×10 5 ~8×10 7 、1×10 5 ~7×10 7 、1×10 5 ~6×10 7 、1×10 5 ~5×10 7 、1×10 5 ~4×10 7 、1×10 5 ~4×10 7 、1×10 5 ~3×10 7 、1×10 5 ~2×10 7 、1×10 5 ~1×10 7 、1×10 5 ~9×10 6 、1×10 5 ~8×10 6 、1×10 5 ~7×10 6 、1×10 5 ~6×10 6 、1×10 5 ~5×10 6 、1×10 5 ~4×10 6 、1×10 5 ~4×10 6 、1×10 5 ~3×10 6 、1×10 5 ~2×10 6 、1×10 5 ~1×10 6 、2×10 5 ~9×10 7 、2×10 5 ~8×10 7 、2×10 5 ~7×10 7 、2×10 5 ~6×10 7 、2×10 5 ~5×10 7 、2×10 5 ~4×10 7 、2×10 5~4×10 7 , 2×10 5 ~3×10 7 , 2×10 5 ~2×10 7 , 2×10 5 ~1×10 7 , 2×10 5 ~9×10 6 , 2×10 5 ~8×10 6 , 2×10 5 ~7×10 6 , 2×10 5 ~6×10 6 , 2×10 5 ~5×10 6 , 2×10 5 ~4×10 6 , 2×10 5 ~4×10 6 , 2×10 5 ~3×10 6 , 2×10 5 ~2×10 6 , 2×10 5 ~1×10 6 , and 3 × 10 5 ~3×10 6 The effective dose may include, but is not limited to, cells / kg, etc. In addition, the dose may be adjusted considering whether a single dose or multiple doses are to be administered. The exact determination of what is to be considered as an effective dose may be based on factors specific to each subject.

[0445] As used herein, the terms "treat", "treating" and "treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer that may, but is not necessarily, be discernible in the subject. The terms "treat", "treating" and "treatment" may also refer to causing regression, prevention of progression, or at least a slowing down of progression of a disease, disorder or condition. In certain embodiments, "treat", "treating" and "treatment" refer to the alleviation, prevention of onset or development, or shortening of the duration of one or more symptoms associated with a disease, disorder or condition, such as a tumor or cancer. In certain embodiments, "treat", "treating" and "treatment" refer to the prevention of recurrence of a disease, disorder or condition. In certain embodiments, "treat", "treating" and "treatment" refer to the prolongation of survival of a subject having a disease, disorder or condition. In certain embodiments, "treat", "treating" and "treatment" refer to the elimination of a disease, disorder or condition.

[0446] The cells and / or pharmaceutical compositions of the present application can be administered in combination with one or more additional therapeutic agents. In certain embodiments, the one or more therapeutic agents are selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or surrogates thereof, vectors comprising one or more polynucleotides of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (ImiDs). EXAMPLES

[0447] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, but not limit, the disclosure.

[0448] [Example 1] Site-specific manipulation of iPSCs Prior to beginning, ensure cells are healthy and approximately 70% confluent before nucleofection. Add H1152 Rock Inhibitor to a final concentration of 1 μM (1:100) and place in the incubator for 1 hour. Add 3 ml of media + 1 μM Rock Inhibitor to the desired number of wells of a 6-well plate and place in the incubator.

[0449] Prepare primary P3 electroporation buffer: ○ 82 μl of nucleofection solution ○ 18μl of supernatant

[0450] Mix.

[0451] In a sterile tube, mix the following RNP components (per sample+1): 1.4 μl PBS ○ 1.6μl of 100μM guide RNA ○ 2 μl MAD7 (60 μM).

[0452] The components are mixed thoroughly, spun down and allowed to incubate at ambient temperature for 15 minutes. The RNPs are placed at 4° C. until the cells are ready to be electroporated.

[0453] Aspirate spent medium from flask, add 7 ml DPBS, vortex gently, and aspirate. Add 2 ml TrypLE and incubate at 37°C for 3-4 minutes. Add 7 ml E8 medium and gently pipette up and down 2-3 times to obtain cells in single cell suspension. Centrifuge at 300 x g for 3 minutes, then aspirate cells and resuspend in 10 ml cold Opti-MEM (4-6 ml if original flask was less confluent). Re-plate some cells before processing. Count cells and transfer 1.5 x 10 cells (per electroporation) to a new sterile tube. 6 Pipette in cells.

[0454] Centrifuge at 300×g for 3 minutes and then resuspend the cells in Primary P3 solution (100 μl per electroporation).

[0455] For each electroporation, add the components listed in Table 7 to a sterile Eppendorf tube.

[0456] [Table 7]

[0457] Mix thoroughly but gently and transfer 100 μl of the above mix into an EP cuvette, avoiding any bubbles. Tap gently and place the cuvette into the nucleofector unit and use program CA-137. After electroporation, remove the cuvette. Using the prepared sterile pipette, transfer the electroporated cells into one well of a 6-well plate containing 3 ml of medium + 1 μM H11252. Place the plate in the incubator and re-feed and check the operation status daily for 7-10 days after electroporation.

[0458] [Example 2] Editing of the B2M locus The HLA-E-2A-HLA-G transgene donor plasmid (pDNA described above) was specifically engineered to insert the HLA-E-2A-HLA-G transgene at the B2M site (Figure 3) (Figure 12B).

[0459] FIG. 10 depicts flow cytometry analysis of engineered iPSCs. Flow cytometry analysis of cells was performed including post-sorting for HLA-E and HLA-G positive cells (FIG. 10, top and bottom panels, respectively). Results show expression of both HLA-E and HLA-G in iPSCs following HDR into the B2M locus of iPSCs using a bicistronic HDR vector. Additional flow cytometry analysis demonstrated expression of both HLA-E and HLA-G engineered by homologous recombination repair (HDR) into iPSCs, enriched for only HLA-E expression (FIG. 11), thus confirming that bicistronic HDR vectors encoding both HLA-E and HLA-G can be used to simultaneously express two transgenes.

[0460] [Example 3] Editing the AAVS1 locus The HLA-E-2A-HLA-G transgene donor plasmid is specifically engineered to insert HLA-E-2A-HLA-G at the AAVS1 site (e.g., FIG. 2). Various flow cytometric analyses of engineered cells are performed. As an example, the expression of HLA-E and HLA-G is determined in iPSCs after HDR to iPSCs.

[0461] [Example 4] Editing the CIITA locus The HLA-E-2A-HLA-G transgene donor plasmid is specifically engineered to insert HLA-E-2A-HLA-G at the CIITA site (e.g., FIG. 4). Various flow cytometric analyses of engineered cells are performed. As an example, the expression of HLA-E and HLA-G is determined in iPSCs after HDR to iPSCs.

[0462] [Example 5] Editing the CLYBL locus HLA-E-2A-HLA-G transgene donor plasmid is specifically engineered to insert HLA-E-2A-HLA-G transgene into CLYBL site. Various flow cytometric analysis of engineered cells is performed. As an example, the expression of HLA-E and HLA-G is determined in iPSCs after HDR to iPSCs.

[0463] [Example 6] Editing the NKG2A locus HLA-E-2A-HLA-G transgene donor plasmid is specifically engineered to insert HLA-E-2A-HLA-G transgene into NKG2A site. Various flow cytometric analysis of engineered cells is performed. As an example, the expression of HLA-E and HLA-G is determined in iPSC after HDR to iPSC.

[0464] [Example 7] HLA-E and HLA-G engineered cells NK cells undergo a balance of activating and inhibitory signals. They recognize the absence of HLA class I as a sign of DNA damage or viral infection and are able to kill target cells. Due to the genetic diversity of HLA class I proteins between populations, these molecules must be removed from iNK to prevent recognition and elimination by the recipient. This assay shows a potent killing of iNK cells by primary NK cells (e.g., isolated vs. PBMC) when they lack HLA I proteins. To overcome this phenomenon, iNK cells were engineered to express HLA-E and HLA-G (SEQ ID NO: 31), which are more conserved in human populations.

[0465] material: Donor blood sample (25-50 × 10 -6 cells / vial; frozen aliquots) ○ iNK cells Cell Trace Yellow (CTY), ThermoFisher Scientific Catalog No. C34567 Cell Trace Violet, ThermoFisher Scientific Catalog No. C34557 CD56 APC, Biolegend Catalog No. 981204 ○ Dead Cell Removal Kit, Miltenyi #130-090-101.

[0466] PBMCs were removed from donor blood samples and counted. PBMCs were resuspended and passed through a 40 μM cell strainer into a 50 mL conical tube. 5 mL of PBS was used to wash the well and cell strainer. This resulted in a total volume of 10 mL of cell suspension.

[0467] PBMC donor cells were stained with CTY according to the following protocol: ○ Cells were washed with 1x PBS. ○ 2 × 10 cells in 1 × PBS 6 The cells were resuspended at a concentration of 1000 cells / mL. o An equal volume of CYT stain was added (1:2000 concentration in 1x PBS). ○ Cells were incubated for 15 minutes at room temperature (RT) away from light. * Stained cells were agitated (e.g., by vortexing or shaking the tube) every 3-5 min to ensure even distribution of the stain. After 15 minutes, medium was added to each tube to double the initial volume. The cell suspension was centrifuged and the supernatant removed from the cell pellet. The cells were resuspended in 10 mL of medium to further wash away any residual CTY. ○ Cell viability was determined. ○ 2.5 × 10 cells in assay medium with the required supplements 6 Resuspended at a final concentration of cells / mL.

[0468] iNK cells were rested overnight in 5 mL NK cell complete medium (NKCM) + 10 ng / mL IL15 and then harvested. Wells were rinsed with an additional 5.2 mL PBS and viability was determined.

[0469] 100 μl of cell suspension was used to check for HLA-E and HLA-G expression. One well of each sample was stained with LIVE / DEAD™ Fixable Near-ID Dead Cell Stain (ThemoFisher #L10119) alone and one with the antibodies listed below.

[0470] Dye Panel: ○ LD-near IR - 1:1000 in 100 μl PBS for 15 min (wash with 150 μl PBS) ○ CD56-BV421 - 1:400 ○ HLA-E-APC - 1:200 HLA-G-PE - 1:200.

[0471] Samples were stained for 15 minutes in 100 μl of BSA staining buffer, washed with 150 μl of PBS, then 200 μl of PBS before fixing with 200 μl of BD™ Stabilizing Color Fixative.

[0472] iNK cells were stained with CTV (15 min, 1 × 10 6 (1:4000 stock diluted in 50 μL DMSO at 10 cells / mL). Cells were resuspended in PBS to 2 × 10 6 Cells / mL and then 2x staining solution was added. Staining was stopped by doubling chromosome volume with 5% FBS RPMI.

[0473] Cell counting after CTV staining: ○ iNK (sample 1) - 0.60×10 6 Cells / mL x 10 mL = 6 x 10 total cells 6 ; Viable cells 58.3% ○ iNK (sample 2) - 0.70×10 6Cells / mL x 10mL = number of cells 7 x 10 6 ; Viable cells 59.7%

[0474] Dead cell removal was performed according to the manufacturer's instructions. The LS column (Miltenyi #130-042-401) was washed with 10 mL of PBS containing FBS. Cell yield after dead cell removal: ○ Sample 1 - 0.47×10 6 Cells / mL x 10 mL; 68.8% viable cells; 2nd count: 0.46 x 10 6 cells / mL x 9.9 mL; 63.9% viable cells => 4.6 x 10 total viable cells 6 ○ Sample 2 - 0.51×10 6 cells / mL x 10.4 mL; 73.3% viable cells => 5.3 x 10 total viable cells 6

[0475] iNK cells were plated in the assay (100 μl or 10 4 For each well, resuspend in NKCM containing 10 ng / mL IL-15. 5 / mL.

[0476] Dead cell removal for the second round (RC01-10) was performed using Miltenyi-supplied binding buffer. 1. Incubate iNK cells with microbeads for 10 min. 6 Cells were resuspended in 10 μl per cell and incubated for 15 minutes at room temperature. 2. 500 μl of 1× binding buffer was added. The cells were passed through a LS column and washed with 3 ml of 1× binding buffer. 3. The column was held on a magnet and washed with approximately 3 mL increments up to a total volume of 10 mL.

[0477] Cell counting before dead cell removal: ○ Sample 1 - 3.86×10 6 Cells / mL; Viable cells 64.1%; Total cells 7.72 x 10 6 ○ Sample 2 - 2.98×10 6Cells / mL; Live cells 63.4%; Total cells 5.96 x 10 6

[0478] Cell counting after removal of dead cells: ○ Sample 1 - Total number of cells: 5.46 x 10 6 ; Viable cells 97.8% ○ Sample 2 - Total number of cells: 4.11 x 10 6 ;96.7% viable cells.

[0479] HLA-E expression on K562 cells provided improved protection from killing over HLA-G, while the combination of both HLA-E and HLA-G conferred improved protection against PBMC cytolysis (Figures 13A-13B). iNK cells edited with HLA-E and HLA-G were consistently protective compared to iNK cells lacking HLA (Figures 14A-14B).

[0480] [Example 8] Expression of HLA-E and HLA-G constructs with different linkers iPSCs were engineered to express HLA-E and HLA-G linked by the linkers (G4S)2 (SEQ ID NO: 165), HLA-E and HLA-G linked by (G4S)3 (SEQ ID NO: 31), and HLA-E and HLA-G linked by (G4S)4 (SEQ ID NO: 168). Expression of HLA-E and HLA-G linked by (G4S)2, (G4S)3, and (G4S)4 from iPSCs was analyzed using flow cytometry. Cells were stained with the following antibodies: HLA-E-APC - 1:200, and HLA-G-PE - 1:200. The results are shown in Figures 15A-15D and 16A-16B. The construct with the (G4S)3 linker (SEQ ID NO: 31) showed the highest expression. * * *

[0481] The present disclosure is not intended to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0482] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety, as if physically presented herein.

[0483] List of Arrays SEQ ID NO: 1 Signal peptide HLA-G amino acid sequence MVVMARPRTLFLLLSGALTLTETWA SEQ ID NO:2 Signal peptide HLA-G nucleotide sequence ATGGTGGTCATGGCCCCTAGAACACTGTTCCTGCTGCTGTCTGGCGCCCTGACACTGACAGAAACATGGGCC SEQ ID NO:3 Signal peptide HLA-G nucleotide sequence ATGGTTGTGATGGCTCCTAGGACTTTGTTTCTGCTGCTCAGCGGCGCTCTGACTCTGACCGAGACTTGGGCT SEQ ID NO: 4: Presented peptide 1 amino acid sequence VMAPRTLIL SEQ ID NO: 5. Presenting peptide 1 nucleotide sequence GTGATGGCCCCACGGACACTGATTCTT SEQ ID NO:6 Linker (G4S)3 amino acid sequence GGGGSGGGGSGGGGS SEQ ID NO:7 Linker (G4S)3 (SEQ ID NO:6) nucleotide sequence GGAGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCT SEQ ID NO:8 Linker (G4S)3 (SEQ ID NO:6) nucleotide sequence GGAGGTGGTGGTAGCGGTGGTGGCGGTTCAGGTGGCGGAGGTTCT SEQ ID NO:9 B2M amino acid sequence IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM SEQ ID NO: 10 B2M nucleotide sequence

[0484] [ka] SEQ ID NO: 11 B2M nucleotide sequence

[0485] [ka] SEQ ID NO: 12 Linker (G4S) 4GS amino acid sequence GGGGSGGGGSGGGGSGGGGSGS SEQ ID NO:13 Linker (G4S)4GS (SEQ ID NO:12) nucleotide sequence GGAGGCGGCGGTAGTGGCGGCGGAGGAAGCGGAGGCGGAGGTTCAGGTGGTGGTGGATCTGGAAGC SEQ ID NO:14 Linker (G4S)4GS (SEQ ID NO:12) nucleotide sequence GGCGGTGGCGGCAGCGGAGGTGGTGGCTCAGGCGGAGGCGGTTCCGGCGGAGGCGGCTCTGGATCT SEQ ID NO: 15 HLA-E amino acid sequence

[0486] [ka] SEQ ID NO: 16 HLA-E nucleotide sequence

[0487] [ka] SEQ ID NO: 17 B2M-HLA-E amino acid sequence

[0488] [ka] SEQ ID NO: 18 B2M-HLA-E nucleotide sequence

[0489] [ka] SEQ ID NO: 19 Signal-presenting-B2M-HLA-E amino acid sequence

[0490] [ka] SEQ ID NO: 20 Signal-presenting-B2M-HLA-E nucleotide sequence

[0491] [ka] SEQ ID NO: 21 P2A amino acid sequence ATNFSLLKQAGDVEENPGP SEQ ID NO:22 P2A nucleotide sequence GCCACCAACTTCAGCCTGCTTAAACAGGCAGGCGACGTGGAAGAGAACCCCGGACCT SEQ ID NO: 23: Presented peptide 2 amino acid sequence RIIPRHLQL SEQ ID NO: 24: Presenting peptide 2 nucleotide sequence AGAATCATCCCCAGACATCTGCAGCTT SEQ ID NO: 25 HLA-G amino acid sequence

[0492] [ka] SEQ ID NO: 26 HLA-G nucleotide sequence

[0493] [ka] SEQ ID NO: 27 B2M-HLA-G amino acid sequence

[0494] [ka] SEQ ID NO: 28 B2M-HLA-G nucleotide sequence

[0495] [ka] SEQ ID NO: 29 Signal-presenting-B2M-HLA-G amino acid sequence

[0496] [ka] SEQ ID NO: 30 Signal-presenting-B2M-HLA-G nucleotide sequence

[0497] [ka] SEQ ID NO: 31 HLA-E-2A-HLA-G transgene amino acid sequence with signal and presentation peptide

[0498] [ka] SEQ ID NO: 32 HLA-E-2A-HLA-G transgene nucleotide sequence with signal and presenting peptide

[0499] [ka] SEQ ID NO:33 Whitlow linker GSTSGSGKPGSGEGSTKG SEQ ID NO:34 Linker 3 GGSEGKSSGSGSESKSTGGS SEQ ID NO:35 Linker 4 GGGSGGGS SEQ ID NO:36 Linker 5 GGGSGGGSGGGS SEQ ID NO:37 Linker 6 GGGSGGGSGGGSGGGS SEQ ID NO:38 Linker 7 GGGSGGGSGGGSGGGSGGGS SEQ ID NO:39 Linker 8(G4S)4 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 40 Linker 9 GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:41 (G4S)2 linker GGGGSGGGGS SEQ ID NO:42(G4S)6 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:43 (G4S)7 linker GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:44 (G4S)8 linker GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:45 Linker 10 IRPRAIGGSKPRVA SEQ ID NO:46 Linker 11 GKGGSGKGGSGKGGS SEQ ID NO:47 Linker 12 GGKGSGGKGSGGKGS SEQ ID NO:48 Linker 13 GGGKSGGGKSGGGKS SEQ ID NO:49 Linker 14 GKGKSGKGKSGKGKS SEQ ID NO:50 Linker 15 GGGKSGGKGSGKGGS SEQ ID NO:51 Linker 16 GKPGSGKPGSGKPGS SEQ ID NO:52 Linker 17 GKPGSGKPGSGKPGSGKPGS SEQ ID NO:53 Linker 18 GKGKSGKGKSGKGKSGKGKS SEQ ID NO:54 Linker 19 STAGDTHLGGEDFD SEQ ID NO:55 Linker 20 GEGGSGEGGSGEGGS SEQ ID NO:56 Linker 21 GGEGSGGEGSGGEGS SEQ ID NO:57 Linker 22 GEGESGEGESGEGES SEQ ID NO:58 Linker 23 GGGESGGEGSGEGGS SEQ ID NO:59 Linker 24 GEGESGEGESGEGESGEGES SEQ ID NO:60 Linker 25 PRGASKSGSASQTGSAPGS SEQ ID NO:61 Linker 26 GTAAAAGAGAAGGAAAGAAG SEQ ID NO:62 Linker 27 GTSGSSGSGSGGSGSGGGG SEQ ID NO:63 Linker 28 G.S.G.S. SEQ ID NO:64 Linker 29 APAPAPAPAP SEQ ID NO:65 Linker 30 APAPAPAPAPAPAPAPAPAP SEQ ID NO:66 Linker 31 AEAAAKEAAAKEAAAAKEAAAAKEAAAAKAAA SEQ ID NO:67 Linker (GGGGS)n, where n is an integer between 1 and 8 SEQ ID NO:68 Hinge EPKSCDKTHTCPPCP SEQ ID NO:69 Hinge ERKCCVECPPCP Sequence number 70 Hinge ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 SEQ ID NO:71 Hinge ESKYGPPCPSCP SEQ ID NO:72 AAVS1 gRNA TTTATCTGTCCCCTCCACCCCACA SEQ ID NO: 73 AAVS1 left homology arm

[0500] [ka] SEQ ID NO: 74 AAVS1 right homology arm

[0501] [ka] SEQ ID NO:75 B2M gRNA TTTACTCACGTCATCCAGCAGAGA SEQ ID NO: 76 B2M left homology arm

[0502] [ka] SEQ ID NO: 77 B2M right homologous arm

[0503] [ka] SEQ ID NO:78 CIITA gRNA TTTACCTTGGGGCTCTGACAGGTA SEQ ID NO:79 CIITA left homology arm

[0504] [ka] SEQ ID NO:80 CIITA right homologous arm

[0505] [ka] SEQ ID NO: 81 CLYBL gRNA AGAGTGATCACAGCTCTGACTAAA SEQ ID NO:82 CLYBL left homology arm

[0506] [ka] SEQ ID NO:83 CLYBL right homology arm

[0507] [ka] SEQ ID NO:84 NKG2A gRNA CTCAGACCTGAATCTGCCCCCAAA SEQ ID NO: 85 NKG2A left homology arm

[0508] [ka] SEQ ID NO: 86 NKG2A right homologous arm

[0509] [ka] SEQ ID NO:87 TRAC gRNA GTGTACCAGCTGAGAGACTCTAAA SEQ ID NO:88 TRAC left homology arm

[0510] [ka] SEQ ID NO:89 TRAC right homology arm

[0511] [ka] SEQ ID NO: 90 HLA-G signal peptide-B2M-HLA-G

[0512] [ka] SEQ ID NO: 91 HLA-G signal peptide-B2M-HLA-G

[0513] [ka] SEQ ID NO: 92 CAG promoter

[0514] [ka] SEQ ID NO:93 SV40 terminator

[0515] [ka] SEQ ID NO: 94 CIITA exon 5 targeting domain sequence TTTCTGCCCAACTTCTGCTGGCAT SEQ ID NO: 95 CIITA exon 5 left homology arm

[0516] [ka] SEQ ID NO: 96 CIITA exon 5 right homology arm

[0517] [ka] SEQ ID NO: 97 CD70 exon 1 targeting domain sequence TTTGGTCCCATTGGTCGCGGGCTT SEQ ID NO: 98 CD70 exon 1 left homology arm

[0518] [ka] SEQ ID NO: 99 CD70 exon 1 right homology arm

[0519] [ka] SEQ ID NO: 100 CD38 exon 1 targeting domain sequence TTTCCCGAGACCGTCCTGGCGCG SEQ ID NO: 101 CD33 exon 5 targeting domain sequence TTTGTCTGCAGGGAAACAAGAGACC SEQ ID NO: 102 CD33 exon 3 targeting domain sequence TTTGGAGTGGCCGGGTTCTAGAGTG SEQ ID NO: 103 WT MAD7 nucleic acid sequence

[0520] [ka]

[0521] [ka] SEQ ID NO: 104 MAD7 codon optimized nucleic acid sequence

[0522] [ka]

[0523] [ka] SEQ ID NO: 105 MAD7 amino acid sequence

[0524] [ka] SEQ ID NO: 106 Scaffold sequence of guide nucleic acid GTTAAGTTATATAGAATAATTTCTACTGTTGTAGA SEQ ID NO: 107 Scaffold sequence of guide nucleic acid CTCTACAACTGATAAAGAATTTCTACTTTTGTAGAT SEQ ID NO: 108 Scaffold sequence of guide nucleic acid GTCTGGCCCCAAATTTTAATTTCTACTGTTGTAGAT SEQ ID NO: 109 gRNA guide sequence specific for the AAVS1 locus UUUAUCUGUCCCCUCCACCCCACA SEQ ID NO: 110 gRNA guide sequence specific for the B2M locus UUUACUCACGUCAUCCAGCAGAGA SEQ ID NO: 111 gRNA guide sequence specific for the CIITA locus UUUACCUUGGGGCUCUGACAGGUA SEQ ID NO: 112 gRNA guide sequence specific for the CITTA exon 5 locus UUUCUGCCCAACUUCUGCUGGCAU SEQ ID NO: 113 gRNA guide sequence specific for the CLYBL locus AGAGUGAUCACAGCUCUGACUAAA SEQ ID NO: 114 gRNA guide sequence specific for the NKG2A locus CUCAGACUGAAUCUGCCCCCAAA SEQ ID NO: 115 gRNA guide sequence specific for the TRAC locus GUGUACCAGCUGAGAGACUCUAAA SEQ ID NO: 116 gRNA guide sequence specific for the CD70 locus UUUGGUCCCAUUGGUCGCGGGCUU SEQ ID NO: 117 gRNA guide sequence specific for the CD38 locus UUUCCCGAGACCGUCCUGGCGCG SEQ ID NO: 118 gRNA guide sequence specific for CD33 exon 5 locus UUUGUCUGCAGGGAAACAAGAGACC SEQ ID NO: 119 gRNA guide sequence specific for CD33 exon 3 locus UUUGGAGUGGCCGGGUUCUAGAGUG SEQ ID NO: 120 HLA-E-2A-HLA-G transgene nucleotide sequence with signal and presenting peptide without terminal A

[0525] [ka] SEQ ID NO: 121 T2A amino acid sequence EGRGSLLTCGDVEENPGP SEQ ID NO:122 Thoseaasigna virus 2A amino acid sequence AEGRGSLLTCGDVEENPGP SEQ ID NO:123 Thoseaasigna virus 2A amino acid sequence GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 124 FMDV2A amino acid sequence GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVESNPGP SEQ ID NO: 125 Sponge 2A amino acid sequence LLCFLLLLLSGDVELNPGP SEQ ID NO: 126 Sponge 2A amino acid sequence HHFMFLLLLLAGDIELNPGP SEQ ID NO: 127 Amino acid sequence of burrowing worm 2A WFLVLLSFILSGDIEVNPGP SEQ ID NO: 128 Amphioxus 2A amino acid sequence KNCAMYMLLLSGDVETNPGP SEQ ID NO: 129 Amphioxus 2A amino acid sequence MVISQLMLKLAGDVEENPGP SEQ ID NO: 130 Porcine teschovirus-1 2A amino acid sequence GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 131 Equine rhinitis A virus 2A amino acid sequence GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 132 2A consensus sequence amino acid sequence DXEX-NPGP SEQ ID NO: 133 GMCSFR signal peptide MLLLVTSLLLCELPHPAFLLIP SEQ ID NO:134 FMC63

[0526] [ka] SEQ ID NO:135 FMC63 VLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT SEQ ID NO: 136 CD28 (AA114-220) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 137 CD3-zeta isoform 3 (AA52-163) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 138 FMC63scFV

[0527] [ka] SEQ ID NO: 139 41BB (AA214-255) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 140 IL2Rb (AA266-551) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 141 CD40 (AA216-277) KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ SEQ ID NO: 142 OX40 (AA236-277) ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 143 CD80 (AA264-288) TYCFAPRCRERRNERLRRESVRPV SEQ ID NO: 144 CD86 (AA269-329) KWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF SEQ ID NO: 145 CD27 (AA213-260) QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO: 146 ICOS (AA162-199) CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL SEQ ID NO: 147 NKG2D (AA1-51) MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENAS SEQ ID NO: 148 DAP10 (AA70-93) LCARPRRSPAQEDGKVYINMPGRG SEQ ID NO: 149 DAP12 (AA62-113) YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK SEQ ID NO: 150 2B4 / CD244 (AA251-370) WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS SEQ ID NO: 151 CD28 (AA180-220) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 152 CD8 (AA136-182) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY SEQ ID NO: 153 CD28 (AA114-151) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP SEQ ID NO: 154 CD8 (AA183-203) IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 155 CD28 (AA153-179) FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 156 Cytoplasmic polyhedrosis virus 2A (BmCPV2A) DVFRSNYDLLKLCGDIESNPGP SEQ ID NO: 157 Flacheria Virus 2A (BmIFV2A) TLTRAKIEDELIRAGIESNPGP SEQ ID NO: 158 CD28 transmembrane domain amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH SEQ ID NO: 159 CD8α transmembrane amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 160 CD3ζ transmembrane amino acid sequence LCYLLDGILFIYGVILTALFL SEQ ID NO: 161 HLA-E transmembrane amino acid sequence VGIIAGLVLLGSVVSGAVVAAVIW SEQ ID NO: 162 HLA-G transmembrane amino acid sequence MGIVAGLVVLAAVVTGAAVAAVLW SEQ ID NO: 163 EGFR transmembrane amino acid sequence IATGMVGALLLLLVVALGIGLFM SEQ ID NO: 165 HLA-E-(G4S)2-HLA-G transgene amino acid sequence with signal and presentation peptide

[0528] [ka] SEQ ID NO: 166 HLA-E-(G4S)2-HLA-G transgene nucleotide sequence with signal and presenting peptide

[0529] [ka] SEQ ID NO: 167 HLA-E-(G4S)3-HLA-G transgene nucleotide sequence with signal and presentation peptide

[0530] [ka] SEQ ID NO: 168 HLA-E-(G4S)4-HLA-G transgene amino acid sequence with signal and presentation peptide

[0531] [ka] SEQ ID NO: 169 HLA-E-(G4S)4-HLA-G transgene nucleotide sequence with signal and presentation peptide

[0532] [ka]

Claims

1. (a) a first molecule comprising an HLA-E heavy chain and (b) a second molecule comprising an HLA-G heavy chain, and (c) a chimeric single chain HLA-E and HLA-G molecule comprising a connecting peptide between (a) and (b).

2. The chimeric single chain HLA-E and HLA-G molecules of claim 1, wherein the order of the chimeric single chain HLA-E and HLA-G molecules is (i)(a)-(c)-(b) or (ii)(b)-(c)-(a).

3. 3. The chimeric single chain HLA-E and HLA-G molecule of claim 1 or claim 2, wherein the HLA-E heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto.

4. 4. The chimeric single chain HLA-E and HLA-G molecule of claim 3, wherein the nucleotide sequence encoding the HLA-E heavy chain polypeptide comprises the sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 80% sequence identity thereto.

5. 5. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 4, wherein the HLA-G heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 80% sequence identity thereto.

6. 6. The chimeric single chain HLA-E and HLA-G molecule of claim 5, wherein the nucleotide sequence encoding the HLA-G heavy chain polypeptide comprises the sequence of SEQ ID NO:26, or a nucleotide sequence having at least 80% sequence identity thereto.

7. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 6, wherein the connecting peptide comprises an autoprotease peptide and optionally one or two autoprotease peptide linkers.

8. 8. The chimeric single chain HLA-E and HLA-G molecule of claim 7, wherein at least one of said autoprotease peptide linkers is 5' to said autoprotease peptide, 3' to said autoprotease peptide, or both 5' and 3' to said autoprotease peptide.

9. 9. The chimeric single chain HLA-E and HLA-G molecule of claim 7 or claim 8, wherein the autoprotease peptide comprises an amino acid sequence shown in Table 3, or an amino acid sequence having at least 80% sequence identity thereto.

10. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 7 to 9, wherein the autoprotease peptide is a 2A peptide.

11. 11. The chimeric single chain HLA-E and HLA-G molecule of claim 10, wherein the 2A peptide is a P2A, F2A, E2A, T2A, GF2A, GP2A, GE2A, GT2A, BmCPV2A, or BmIFV2A peptide.

12. The chimeric single chain HLA-E and HLA-G molecule of claim 10 or claim 11, wherein the 2A peptide is a P2A peptide.

13. 13. The chimeric single chain HLA-E and HLA-G molecule of claim 11 or claim 12, wherein the P2A peptide comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 80% sequence identity thereto.

14. 14. The chimeric single chain HLA-E and HLA-G molecule of claim 13, wherein the nucleotide sequence encoding the P2A peptide comprises the sequence of SEQ ID NO:22, or a nucleotide sequence having at least 80% sequence identity thereto.

15. 15. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 14, wherein (a) the first molecule comprises a first B2M polypeptide fused to the HLA-E heavy chain via a first linker, and / or (b) the second molecule comprises a second B2M polypeptide fused to the HLA-G heavy chain via a second linker.

16. 16. The chimeric single chain HLA-E and HLA-G molecule of claim 15, wherein the first B2M polypeptide is 5' to the HLA-E heavy chain polypeptide.

17. 16. The chimeric single chain HLA-E and HLA-G molecule of claim 15, wherein the first B2M polypeptide is 3' to the HLA-E heavy chain polypeptide.

18. 18. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 15 to 17, wherein the second B2M polypeptide is 5' to the HLA-G heavy chain polypeptide.

19. 18. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 15 to 17, wherein the second B2M polypeptide is 3' to the HLA-G heavy chain polypeptide.

20. 20. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 15 to 19, wherein the first B2M polypeptide and / or the second B2M polypeptide comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 80% sequence identity thereto.

21. 21. The chimeric single chain HLA-E and HLA-G molecule of claim 20, wherein the polynucleotide sequence encoding the first B2M polypeptide and / or the second B2M polypeptide comprises a nucleotide sequence of SEQ ID NO: 10 or 11, or a nucleotide sequence having at least 80% sequence identity thereto.

22. 22. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 21, wherein (a) the first molecule further comprises a first presenting peptide fused to the first B2M polypeptide via a third linker, and / or (b) the second molecule further comprises a second presenting peptide fused to the second B2M polypeptide via a fourth linker.

23. 23. The chimeric single chain HLA-E and HLA-G molecule of claim 22, wherein (a) the first presenting peptide is fused to the first B2M polypeptide and (a) the second presenting peptide is fused to the second B2M polypeptide.

24. 24. The chimeric single chain HLA-E and HLA-G molecule of claim 22 or claim 23, wherein the first and / or second presenting peptides are the same.

25. 24. The chimeric single chain HLA-E and HLA-G molecule of claim 22 or claim 23, wherein the first and / or second presented peptides are different.

26. 26. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 22 to 25, wherein the first presenting peptide and / or the second presenting peptide comprises an amino acid sequence of SEQ ID NO: 4 or 23, or an amino acid sequence having at least 80% sequence identity thereto.

27. The chimeric single chain HLA-E and HLA-G molecule of claim 26, wherein the polynucleotide sequence encoding the first presenting peptide and / or the second presenting peptide comprises a nucleotide sequence of SEQ ID NO: 5 or 24, or a nucleotide sequence having at least 80% sequence identity thereto.

28. 28. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 22 to 27, wherein the first, second, third, fourth, and / or autoprotease peptide linkers each separately comprise an amino acid sequence set forth in Table 4, or an amino acid sequence having at least 80% sequence identity thereto.

29. 29. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 22 to 28, wherein the first peptide linker sequence and / or the second peptide linker sequence comprises an amino acid sequence of SEQ ID NO: 6, 39 or 41, or an amino acid sequence having at least 80% sequence identity thereto.

30. 30. The chimeric single chain HLA-E and HLA-G molecule of claim 29, wherein the nucleotide sequence encoding the first peptide linker sequence and / or the second peptide linker sequence comprises a sequence of SEQ ID NO: 7 or 8, or a nucleotide sequence having at least 80% sequence identity thereto.

31. 31. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 22 to 30, wherein the third peptide linker sequence and / or the fourth peptide linker sequence comprises an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity thereto.

32. 32. The chimeric single chain HLA-E and HLA-G molecule of claim 31 , wherein the nucleotide sequence encoding the third peptide linker sequence and / or the fourth peptide linker sequence comprises a sequence of SEQ ID NO: 13 or 14, or a nucleotide sequence having at least 80% sequence identity thereto.

33. 33. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 32, wherein (a) the first molecule further comprises a first signal peptide operably linked to the HLA-E heavy chain, and / or (b) the second molecule further comprises a second signal peptide operably linked to the HLA-G heavy chain.

34. 34. The chimeric single chain HLA-E and HLA-G molecule of claim 33, wherein said first signal peptide and said second signal peptide are the same.

35. 34. The chimeric single chain HLA-E and HLA-G molecule of claim 33, wherein said first signal peptide and said second presenting peptide are different.

36. 36. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 33 to 35, wherein the first signal peptide and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity thereto.

37. 36. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 33 to 35, wherein the first signal peptide and the second signal peptide comprise the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto.

38. 38. The chimeric single chain HLA-E and HLA-G molecule of claim 36 or claim 37, wherein the polynucleotide sequence encoding the first signal peptide and / or the second signal peptide comprises a polynucleotide sequence of SEQ ID NO: 2 or 3, or a polynucleotide sequence having at least 80% sequence identity thereto.

39. 39. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 38, wherein the first molecule comprises an amino acid sequence of SEQ ID NO: 17 or 19, or an amino acid sequence having at least 80% sequence identity thereto.

40. 40. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 39, wherein the second molecule comprises an amino acid sequence of SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity thereto.

41. 41. The chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 40, comprising the amino acid sequence of SEQ ID NO: 31, 165 or 168.

42. A polynucleotide encoding the chimeric single chain HLA-E and HLA-G molecule of any one of claims 1 to 41.

43. 43. The polynucleotide of claim 42, wherein the polynucleotide sequence encoding the first molecule comprises the nucleotide sequence of SEQ ID NO: 18 or 20, or a nucleotide sequence having at least 80% sequence identity thereto.

44. 44. The polynucleotide of claim 42 or claim 43, wherein the polynucleotide sequence encoding the second molecule comprises the nucleotide sequence of SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity thereto.

45. 45. The polynucleotide of any one of claims 42 to 44, wherein the polynucleotide encoding the single chain HLA-E and HLA-G molecules comprises the nucleotide sequence of SEQ ID NO: 32, 120, 166, 167 or 169.

46. 46. ​​The polynucleotide of any one of claims 42 to 45, wherein the polynucleotide sequences encoding the single chain HLA-E and HLA-G molecules are operably linked to a single promoter.

47. 47. The polynucleotide of claim 46, wherein the promoter is an inducible promoter.

48. 48. A polynucleotide according to any one of claims 42 to 47 which is a DNA molecule.

49. 48. The polynucleotide of any one of claims 42 to 47, which is an RNA molecule.

50. A recombinant vector comprising the polynucleotide according to any one of claims 42 to 49.

51. The recombinant vector of claim 50, which is a viral vector.

52. 52. The recombinant vector of claim 51, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a herpes viral vector, a baculoviral vector, or a vaccinia viral vector.

53. The recombinant vector of claim 50, which is a non-viral vector.

54. 54. The recombinant vector of claim 53, wherein the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a piggyBac transposon, or a single- or double-stranded DNA molecule used as a template for homology-directed repair (HDR)-based gene editing.

55. 55. An isolated host cell comprising a polynucleotide according to any one of claims 42 to 49 or a recombinant vector according to any one of claims 50 to 54.

56. 50. An isolated host cell comprising a chimeric single chain HLA-E and HLA-G molecule encoded by the polynucleotide of any one of claims 42 to 49.

57. 57. The isolated host cell of claim 55 or claim 56, which is an induced pluripotent stem cell (iPSC).

58. 58. The isolated host cell of any one of claims 55 to 57, wherein the host cell is an immune effector cell.

59. 58. An immune effector cell or population thereof derived from the iPSC of claim 57.

60. 60. The immune effector cell of claim 58 or claim 59, which is a T cell, a natural killer (NK) cell, a natural killer T cell (NKT cell), a mesenchymal stem cell (MSC), or a macrophage.

61. The immune effector cell of claim 60, which is a T cell.

62. αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, memory T stem cells (T ＳＣＭ ), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

63. The immune effector cell of claim 60, which is a NK cell.

64. A MAD7 / gRNA ribonucleoprotein (RNP) complex composition for insertion of HLA-E and HLA-G transgenes, comprising: (I) a MAD7 nuclease; (II) a guide RNA (gRNA) specific for said MAD7 nuclease, said gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, said guide sequence being selected from SEQ ID NOs: 109-119, and wherein when said gRNA is complexed with MAD7 nuclease, said guide sequence directs sequence-specific binding of said MAD7 nuclease to said target sequence.

50. The MAD7 / gRNA ribonucleoprotein (RNP) complex composition comprising: a gRNA specific for the MAD7 nuclease; and (III) a transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of the target sequence of the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus, (2) a promoter, (3) a polynucleotide encoding the HLA-E and HLA-G transgenes comprising the polynucleotide of any one of claims 42-49 to which the promoter is operably linked, and (4) a transcription terminator sequence.

65. 1. A MAD7 / gRNA ribonucleoprotein (RNP) complex composition for insertion of HLA-E and HLA-G transgenes, comprising: I) a MAD7 nuclease system, encoded by one or more vectors, said one or more vectors comprising: (a) a sequence encoding a guide RNA (gRNA) operably linked to a first regulatory element, said gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, or CLYBL locus in a cell, said guide sequence being selected from SEQ ID NOs: 109-119, and wherein when transcribed, said guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; (b) a MAD7 nuclease system encoding a MAD7 nuclease system; and (II) an HLA-E and HLA-G transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of the target sequence of the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter; (3) a polynucleotide encoding the HLA-E and HLA-G transgene comprising the polynucleotide of any one of claims 42-49 to which the promoter is operably linked; and (4) a transcription terminator sequence.

66. A MAD7 / gRNA ribonucleoprotein (RNP)-based vector system, comprising: (I) one or more vectors comprising: (a) a sequence encoding a guide RNA (gRNA), said sequence operably linked to a first regulatory element, said gRNA comprising a guide sequence capable of hybridizing to a target sequence at an AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus in a cell, said gRNA guide sequence being selected from SEQ ID NOs: 109-119, wherein when transcribed, said guide sequence directs sequence-specific binding of the MAD7 complex to the target sequence; (b) a sequence encoding a MAD7 nuclease, said sequence operably linked to a second regulatory element; and (II) an HLA-E and HLA-G transgene vector comprising: (1) left and right polynucleotide sequences homologous to left and right arms of the target sequence of the AAVS1, B2M, CIITA, NKG2A, TRAC, CD70, CD38, CD33, or CLYBL locus; (2) a promoter; (3) a polynucleotide encoding an HLA-E and HLA-G transgene comprising the polynucleotide of any one of claims 42-49 to which the promoter is operably linked; and (4) a transcription terminator sequence.

67. 67. The composition of claim 64 or claim 65 or the vector system of claim 66, wherein the cell is an induced pluripotent stem cell (iPSC).

68. 68. A composition according to any one of claims 64, 65 or 67 or a vector system according to claim 66 or 67, wherein the first and / or second regulatory element is a promoter.

69. 69. A composition described in any one of claims 64, 65, 67 or 68, or a vector system described in any one of claims 66 to 68, wherein the first regulatory element and the second regulatory element are the same.

70. 70. A composition described in any one of claims 64, 65, or 67 to 69, or a vector system described in any one of claims 66 to 69, wherein the first regulatory element and the second regulatory element are different.

71. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for the AAVS1 locus.

72. 72. The composition or vector system of Claim 71, wherein the gRNA guide sequence comprises SEQ ID NO:

109.

73. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for a B2M locus.

74. 74. The composition or vector system of Claim 73, wherein the gRNA guide sequence comprises SEQ ID NO:

110.

75. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for the CIITA locus.

76. 76. The composition or vector system of Claim 75, wherein the gRNA guide sequence comprises SEQ ID NO: 111 or 112.

77. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for the NKG2A locus.

78. 78. The composition or vector system of Claim 77, wherein the gRNA guide sequence comprises SEQ ID NO:

114.

79. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for a TRAC locus.

80. 80. The composition or vector system of Claim 79, wherein the gRNA guide sequence comprises SEQ ID NO:

115.

81. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for the CD70 locus.

82. 82. The composition or vector system of Claim 81, wherein the gRNA guide sequence comprises SEQ ID NO:

116.

83. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for the CD38 locus.

84. 84. The composition or vector system of Claim 83, wherein the gRNA guide sequence comprises SEQ ID NO:

117.

85. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-70, wherein the gRNA guide sequence is specific for the CD33 locus.

86. 86. The composition or vector system of Claim 85, wherein the gRNA guide sequence is specific for the CD33 locus and comprises SEQ ID NO: 118 or 119.

87. 71. The composition of any one of claims 64, 65, or 67-70, or the vector system of any one of claims 66-67, wherein the gRNA guide sequence is specific for a CLYBL locus.

88. 88. The composition or vector system of Claim 87, wherein the gRNA guide sequence comprises SEQ ID NO:

113.

89. 73. The composition of any one of claims 64, 65, or 67-72, or the vector system of any one of claims 66-72, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of AAVS1 comprise the nucleotide sequences of SEQ ID NOs: 73 and 74, respectively, or fragments thereof.

90. 75. The composition of any one of claims 64, 65, 67-70, 73 or 74, or the vector system of any one of claims 66-70, 73 or 74, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of B2M comprise the nucleotide sequences of SEQ ID NOs: 76 and 77, respectively, or fragments thereof.

91. The composition of any one of claims 64, 65, 67 to 70, 75 or 76, or the vector system of any one of claims 66 to 70, 75 or 76, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of the CIITA comprise (i) the nucleotide sequences of SEQ ID NOs: 79 and 80, respectively, or (ii) SEQ ID NOs: 95 and 96, respectively, or fragments thereof.

92. The composition of any one of claims 64, 65, 67-70, 77 or 78, or the vector system of any one of claims 66-70, 77 or 78, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of NKG2A comprise the nucleotide sequences of SEQ ID NOs: 85 and 86, respectively, or fragments thereof.

93. The composition of any one of claims 64, 65, 67-70, 79 or 80, or the vector system of any one of claims 66-70, 79 or 80, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of the TRAC comprise the nucleotide sequences of SEQ ID NOs: 88 and 89, respectively, or fragments thereof.

94. The composition of any one of claims 64, 65, 67-70, 80 or 82, or the vector system of any one of claims 66-70, 81 or 82, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CD70 comprise nucleotide sequences of SEQ ID NOs: 98 and 99, respectively, or fragments thereof.

95. 89. The composition of any one of claims 64, 65, 67-70, 87 or 88, or the vector system of any one of claims 66-70, 87 or 88, wherein the left and right polynucleotide sequences homologous to the left and right arms of the target sequence of CLYBL comprise the nucleotide sequences of SEQ ID NOs: 82 and 83, respectively, or a fragment thereof.

96. 96. The composition of any one of claims 64, 65, or 67-95, or the vector system of any one of claims 66-95, wherein expression of an endogenous gene comprising a target sequence complementary to the guide sequence of the gRNA molecule is reduced or eliminated in the cell when the RNP complex is introduced into the cell.

97. 97. One or more retroviruses constituting the vector system of any one of claims 66 to 96.

98. 98. An isolated host cell transformed by the vector system of any one of claims 66 to 96 or one or more retroviruses of claim 97.

99. 99. The isolated host cell of claim 98, which is an iPSC.

100. 100. The isolated host cell of claim 98 or claim 99, which is an immune effector cell.

101. 100. An immune effector cell or population thereof derived from an iPSC according to claim 99.

102. The immune effector cell of claim 100 or claim 101, which is a T cell, a natural killer (NK) cell, a natural killer T cell (NKT cell), a mesenchymal stem cell (MSC), or a macrophage.

103. The immune effector cell of claim 102, which is a T cell.

104. αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, memory T stem cells (T ＳＣＭ ), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

105. The immune effector cell of claim 102, which is a NK cell.

106. 106. An immune effector cell according to any of claims 58 to 63 or 100 to 105, which exerts an improved protective effect against allogeneic cell lysis compared to a control cell that does not express said chimeric single chain HLA-E and HLA-G molecules.

107. 107. A pharmaceutical composition comprising an isolated host cell or an iPSC-derived immune effector cell according to any one of claims 55-63 or 98-105.

108. 107. A method for preventing or treating cancer, comprising the step of administering to an individual in need thereof a therapeutically effective amount of a host cell, immune effector cell or said population of claims 55-63 or 98-105, or a pharmaceutical composition of claim 107.

109. 109. The method of claim 108, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, liver cancer, melanoma, bone cancer, breast cancer, colon cancer, leukemia, uterine cancer, ovarian cancer, lymphoma, and brain cancer.

110. 110. The method of treatment of claim 109, wherein the cancer is selected from the group consisting of leukemias, e.g., AML, CML, ALL and CLL, lymphomas, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma, and solid cancers, e.g., sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and biliary tract cancer, thyroid cancer, thymic cancer, bone cancer, and brain cancer, and cancer of unknown etiology (CUP).

111. 111. The method of claim 110, wherein the individual has minimal residual disease (MRD) after an initial cancer treatment.

112. 111. The method of claim 110, wherein the individual has no minimal residual disease (MRD) after one or more cancer treatments or repeat administrations.

113. 10. A method for protecting immune effector cells from allogeneic cell lysis, comprising the step of introducing into said immune effector a polynucleotide described in any one of claims 42 to 49, a recombinant vector described in any one of claims 50 to 54, or a composition described in any one of claims 64, 65, or 67 to 96, or a vector system described in any one of claims 66 to 96.

114. 114. The method of claim 113, wherein the immune effector cell is a T cell, a natural killer (NK) cell, a natural killer T cell (NKT cell), a mesenchymal stem cell (MSC), or a macrophage.

115. 115. The method of claim 114, wherein the immune effector cell is a T cell.

116. αβ T cell receptor (TCR) T cells, γδ T cells, CD8+ T cells, CD4+ T cells, cytotoxic T cells, invariant natural killer T (iNKT) cells, memory T cells, memory T stem cells (T ＳＣＭ ), a naive T cell, an effector T cell, a T helper cell, or a regulatory T cell (Treg).

117. The immune effector cell of claim 114, which is a NK cell.

118. 118. An immune effector cell according to any one of claims 114 to 117, which is derived from an iPSC.

Images