CD16 variants and genetically engineered cells expressing NKG2D and their use

Abstract

CD16 variants and engineered iPSCs expressing NKG2D and cells derived therefrom are disclosed herein. The engineered iPSCs and cells derived therefrom can also express a chimeric antigen receptor. Furthermore, polynucleotide constructs useful for expressing CD16 variants and NKG2D in such cells and methods of using the same are provided herein.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of priority of U.S. Provisional Application No. 63 / 350,298, filed on June 8, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation of Sequence Listing This application was electronically filed in XML format in accordance with WIPO STANDARD ST.26 and contains a sequence listing that is incorporated herein by reference in its entirety. The name of the XML copy created on June 7, 2023 is "SL.xml" and the size is 150,741 bytes.

Background Art

[0003] Cancer remains a major global health problem despite considerable research efforts and scientific progress in treating the disease. Cancer immunotherapy is desirable because it is highly specific and can facilitate the elimination of cancer cells by using the patient's immune system. Research continues to develop effective immune cell-based therapies for treating cancer.

[0004] Activated NK cells can kill target cells such as cancer cells by means similar to those of cytotoxic T cells (i.e., via cytolytic granules containing perforin and granzymes and via the cell death receptor pathway). Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines that promote the recruitment of other white blood cells to target tissues such as cancer tissue.

[0005] Sources of immune cells include those differentiated from induced pluripotent stem cells (iPSCs). These cells can be modified to be allogeneic. There remains an unmet need for therapeutically sufficient and functional engineered allogeneic iPSC-derived therapies for treating cancer. Furthermore, in engineering cell therapies, it is desirable to minimize the number of gene edits that need to be made to the cells. Accordingly, there is a need for engineered cell therapies with multiple functions that can be engineered using a minimal number of edits.

Summary of the Invention

[0006] In one aspect, an induced pluripotent stem cell (iPSC) or a derivative cell thereof is described that includes an exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-protease peptide.

[0007] In some embodiments, the CD16 protein is a CD16 variant protein. In some embodiments, the CD16 variant is a high-affinity CD16 variant. In many embodiments, the CD16 variant is a non-cleavable CD16 variant. In various embodiments, the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2 and 5.

[0008] In some embodiments, the NKG2D protein is a wild-type NKG2D protein. In many embodiments, the NKG2D protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

[0009] In some embodiments, the self-protease peptide is selected from the group consisting of tesehovirus-1 2A (P2A) peptide, foot-and-mouth disease virus 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus 2A (T2A) peptide, cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and BmIFV2A peptide. In many embodiments, the self-protease peptide is a P2A peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3.

[0010] In some embodiments, the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6.

[0011] In some embodiments, the exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPBP locus, NLRC5 locus, RFXANK locus, RFX5 locus, RFXAP locus, CISH locus, CBLB locus, SOCS2 locus, PD1 locus, CTLA4 locus, LAG3 locus, TIM3 locus, and TIGIT locus.

[0012] In some embodiments, the exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, TAP1 locus, TAP2 locus, TAPBP locus, TRAC locus, TRBC1 locus, RFXANK locus, RFX5 locus, and RFXAP locus, whereby the expression of the gene is disrupted.

[0013] In some embodiments, the exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CD70 locus, the CLYBL locus, the NKG2A locus, and the TRAC locus, whereby the expression of the gene is disrupted.

[0014] In some embodiments, the disruption of the gene includes the elimination or reduction of the expression of the gene. In some embodiments, the integration into the gene locus is effected by targeted genome editing.

[0015] In some embodiments, targeted genome editing includes using a method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variants thereof.

[0016] In some embodiments, the method further includes the disruption of one or more genes selected from the group consisting of the AAVS1 gene, the B2M gene, the CIITA gene, the CD70 gene, the CLYBL gene, the NKG2A gene, the NKG2D gene, the TAP1 gene, the TAP2 gene, the TAPBP gene, the TRAC gene, the TRBC1 gene, the RFXANK gene, the RFX5 gene, the RFXAP gene, and any combination thereof.

[0017] In some embodiments, the disruption is the disruption of the B2M gene and the CIITA gene.

[0018] In some embodiments, the iPSC or a derivative cell thereof according to claim 18 or 19, wherein the disruption of the one or more genes includes the elimination or reduction of the expression of the one or more genes. In many embodiments, the disruption of the one or more genes is effected by targeted genome editing.

[0019] In some embodiments, the targeted genome editing involves using a method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variants thereof.

[0020] In some embodiments, the iPSC or the derived cell further comprises a second exogenous polynucleotide encoding an IL-15 protein. In some embodiments, the IL-15 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16.

[0021] In some embodiments, the iPSC or the derived cell further comprises a second exogenous polynucleotide encoding a fusion polypeptide comprising IL-15 and interleukin-15 receptor alpha (IL-15Rα). In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 17.

[0022] In some embodiments, the iPSC or the derived cell further comprises a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) protein. In some embodiments, HLA-E comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14.

[0023] In some embodiments, the iPSC or the derived cell further comprises a fourth exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G) protein. In some embodiments, HLA-G comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15.

[0024] In some embodiments, the HLA-E protein and the HLA-G protein are operably linked by a second self-protease peptide. In some embodiments, the second self-protease peptide is selected from the group consisting of a P2A peptide, an F2A peptide, an E2A peptide, a T2A peptide, a BmCPV2A peptide, and a BmIFV2A peptide.

[0025] In some embodiments, the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus, and any combination thereof.

[0026] In some embodiments, the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the TRAC locus, the TRBC1 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, and any combination thereof.

[0027] In some embodiments, the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, TAP1 locus, TAP2 locus, TAPBP locus, TRAC locus, TRBC1 locus, RFXANK locus, RFX5 locus, RFXAP locus, and any combination thereof, whereby one or more genes are disrupted. In some embodiments, the disruption in one or more genes comprises elimination or reduction of expression of one or more genes.

[0028] In some embodiments, the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, TRAC locus, and any combination thereof, whereby one or more genes are disrupted. In some embodiments, the disruption in one or more genes comprises elimination or reduction of expression of one or more genes.

[0029] In some embodiments, the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMC). In some embodiments, the iPSC is derived from reprogrammed NK or T cells.

[0030] In some embodiments, the iPSC or its derivative cells further comprise a fifth exogenous polynucleotide encoding a chimeric antigen receptor (CAR) that binds to a target antigen.

[0031] In some embodiments, the target antigen is selected from the group consisting of 17-1A antigen, A3, A33 antigen, AFP, B7H4, Ba733, BCMA, BrE3 antigen, CA125, CA9 (CAIX), CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD22, CD23, CD25, CD30, CD33, CD33, CD38, CD45, CD70, CD74, CD79, CD79a, CD80, CD123, CD133, CD138, CEACAM5, CEACAM6, CLDN18.2, CLL1, cMET, colon-specific antigen-p (CSAp), ED-B fibronectin, EGFR, EGFRvIII, EGP-1, EGP-2, EpCAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FGFR1, FGFR3, Flt-1, Flt-3, FOLR1, FOLR2, FOLR3, FSHR, GD2, GPC-3, GPRC5D, HCG, HCG subunit, HER2, HIF-I, HLA-DR, Ia, IGF-I, IL13Rα2, IL-2, IL-6, IL-8, KC4 antigen, KS-1 antigen, KS1-4 antigen, Le-Y, MAGE, MET, MIF, MSLN, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA90, NCA95, nectin-4, p53, PAP, PDGFRA, PLGF, PSA, PSMA, ROBO1, RS5, S100, SLAM F7, SLITRK6, TAC, TAG-72, tenascin-C, tenascin-R, tenascin-W, tenascin-X, Thomson-Friedenreich antigen, Tn antigen, TRAILR1, TRAILR2, TRAILR3, TRAILR4, VEGF, tumor necrosis antigen, angiogenesis antigen, and oncogene antigen. In some embodiments, the CAR comprises an antigen-binding domain selected from the group consisting of any of those provided in Tables 1, 2, and 3.

[0032] In some embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to a target antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) one or more co-stimulatory domains. In some embodiments, the signal peptide of the CAR comprises a GMCSFR signal peptide. In some embodiments, the extracellular domain of the CAR comprises a single-chain Fv (scFv) or VHH domain that specifically binds to a target antigen. In some embodiments, the hinge region of the CAR comprises a CD28 hinge region. In some embodiments, the transmembrane domain of the CAR comprises a CD28 transmembrane domain. In some embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ intracellular domain. In some embodiments, one or more co-stimulatory domains of the CAR comprise a CD28 signaling domain.

[0033] In some embodiments, the derived cell is an NK cell or a T cell. In other embodiments, the derived cell is an NK cell. In many embodiments, the derived cell is a T cell. In some embodiments, the derived cell is a CD34+ hematopoietic progenitor cell.

[0034] In some embodiments, provided is a composition comprising any one population of iPSCs or their derived cells described herein.

[0035] In another aspect, provided is an engineered cell comprising: (i) a first exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-cleaving peptide; (ii) a second exogenous polynucleotide encoding an exogenous polypeptide comprising an IL-15 protein; and (iii) optionally, a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or a fourth exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G).

[0036] In some embodiments, the engineered cell further comprises a fifth polynucleotide encoding a combined artificial cell death / reporter system polypeptide comprising an intracellular domain having herpes simplex virus thymidine kinase (HSV-TK) and a linker, a transmembrane region, and an extracellular domain comprising a prostate-specific membrane antigen (PSMA) extracellular domain or a fragment thereof.

[0037] In some embodiments, HSV-TK comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23 or 29.

[0038] In some embodiments, the combined artificial cell death / reporter system polypeptide comprises HSV-TK fused via a linker to a truncated mutant PSMA polypeptide.

[0039] In some embodiments, the truncated mutant PSMA polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24.

[0040] In some embodiments, the linker comprises a self-cleaving protease peptide sequence selected from the group consisting of P2A peptide sequence, T2A peptide sequence, E2A peptide sequence, and F2A peptide sequence. In certain embodiments, the linker is selected from any one of the group consisting of those described in Table 4.

[0041] In some embodiments, the artificial cell death / reporter system polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 25.

[0042] In some embodiments, the artificial cell death / reporter system polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27, 30, and 31.

[0043] In some embodiments, the artificial cell death / reporter system polypeptide comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 26, 28, and 32.

[0044] In another aspect, there is provided an engineered cell comprising: (i) a first exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-cleaving protease peptide; (ii) a second exogenous polynucleotide encoding a fusion polypeptide comprising an IL-15 protein and an IL-15 receptor alpha (IL-15Rα) protein; and (iii) optionally, a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) protein and / or a fourth exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G) protein.

[0045] The engineered cell is an engineered induced pluripotent stem cell (iPSC), an engineered natural killer (NK) cell, or an engineered T cell.

[0046] In some embodiments, the first exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 6.

[0047] In some embodiments, the second exogenous polynucleotide comprises a nucleic acid sequence encoding the IL-15 / IL-15Rα fusion protein of SEQ ID NO: 17. In some embodiments, the second exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the second exogenous polynucleotide comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the IL-15 protein comprises the amino acid sequence of SEQ ID NO: 16.

[0048] In some embodiments, the third exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 21, and the fourth exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 22.

[0049] In some embodiments, the HLA-E protein and the HLA-G protein are linked by a self-protease peptide. In some embodiments, the HLA-E protein of the engineered cell comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the HLA-G protein of the engineered cell comprises the amino acid sequence of SEQ ID NO: 20. The nucleic acid sequence of SEQ ID NO: 21 encoding the amino acid sequence of SEQ ID NO: 19 is described herein. The nucleic acid sequence of SEQ ID NO: 22 encoding the amino acid sequence of SEQ ID NO: 20 is described herein.

[0050] In some embodiments, the engineered cell further comprises disruption of the B2M and CIITA genes. In some embodiments, the disruption of the B2M and CIITA genes is caused by targeted genome editing. In some embodiments, the targeted genome editing comprises using a method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variations thereof.

[0051] In some embodiments, the first exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.

[0052] In some embodiments, the second exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the collagen locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.

[0053] In some embodiments, the third exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the collagen locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.

[0054] In some embodiments, the fourth exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the collagen locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.

[0055] In some embodiments, the first exogenous polynucleotide and either the second, third, or fourth exogenous polynucleotide are integrated within the B2M gene locus and the CIITA gene locus, thereby disrupting the B2M and CIITA genes. In certain embodiments, the first exogenous polynucleotide is integrated within the CD70 locus and the second exogenous polynucleotide is integrated within the B2M gene locus, thereby disrupting the CD70 and B2M genes. In various embodiments, the first exogenous polynucleotide is integrated within the CD70 locus and the second exogenous polynucleotide is integrated within the CIITA gene locus, thereby disrupting the CD70 and CIITA genes.

[0056] In some embodiments, the first exogenous polynucleotide is integrated within the CD70 locus and the third or fourth exogenous polynucleotide is integrated within the B2M gene locus, thereby disrupting the CD70 and B2M genes.

[0057] In many embodiments, the first exogenous polynucleotide is integrated within the CD70 locus and the third or fourth exogenous polynucleotide is integrated within the CIITA gene locus, thereby disrupting the CD70 and CIITA genes. In various embodiments, the integration into the CD70 locus is into exon 1 of the CD70 gene.

[0058] In some embodiments, integration into the genetic locus occurs by targeted genome editing. In many embodiments, targeted genome editing involves using a method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variations thereof.

[0059] In some embodiments, the engineered cell further comprises a fifth exogenous polynucleotide encoding a chimeric antigen receptor (CAR) that binds to a target antigen. In many embodiments, the target antigen is selected from the group consisting of 17-1A antigen, A3, A33 antigen, AFP, B7H4, Ba733, BCMA, BrE3 antigen, CA125, CA9 (CAIX), CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD22, CD23, CD25, CD30, CD33, CD33, CD38, CD45, CD70, CD74, CD79, CD79a, CD80, CD123, CD133, CD138, CEACAM5, CEACAM6, CLDN18.2, CLL1, cMET, colon-specific antigen-p (CSAp), ED-B fibronectin, EGFR, EGFRvIII, EGP-1, EGP-2, EpCAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FGFR1, FGFR3, Flt-1, Flt-3, FOLR1, FOLR2, FOLR3, FSHR, GD2, GPC-3, GPRC5D, HCG, HCG subunit, HER2, HIF-I, HLA-DR, Ia, IGF-I, IL13Rα2, IL-2, IL-6, IL-8, KC4 antigen, KS-1 antigen, KS1-4 antigen, Le-Y, MAGE, MET, MIF, MSLN, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA90, NCA95, nectin-4, p53, PAP, PDGFRA, PLGF, PSA, PSMA, ROBO1, RS5, S100, SLAM F7, SLITRK6, TAC, TAG-72, tenascin-C, tenascin-R, tenascin-W, tenascin-X, Thomson-Friedenreich antigen, Tn antigen, TRAILR1, TRAILR2, TRAILR3, TRAILR4, VEGF, tumor necrosis antigen, angiogenesis antigen, and oncogene antigen. In some embodiments, the CAR comprises an antigen-binding domain selected from the group consisting of any of those provided in Tables 1, 2, and 3.

[0060] In some embodiments, the CAR comprises: (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to a target antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) one or more co-stimulatory domains.

[0061] In certain embodiments, the signal peptide of the CAR comprises the GMCSFR signal peptide. In many embodiments, the extracellular domain comprises a single-chain Fv (scFv) or VHH domain that specifically binds to a target antigen. In various embodiments, the hinge region comprises the CD28 hinge region. In some embodiments, the transmembrane domain comprises the CD28 transmembrane domain. In certain embodiments, the intracellular signaling domain comprises the CD3ζ intracellular domain. In some embodiments, the one or more co-stimulatory domains comprise the CD28 signaling domain. In some embodiments, the engineered iPSC is differentiated into an engineered differentiated cell.

[0062] In some embodiments, the engineered iPSC is differentiated into an engineered NK cell. In some embodiments, the engineered iPSC is differentiated into an engineered T cell. In some embodiments, the engineered iPSC is differentiated into an engineered CD34+ hematopoietic progenitor cell.

[0063] Provided are compositions comprising any one population of the engineered iPSCs described herein. Provided are compositions comprising any one population of the engineered differentiated cells described herein. Also provided are compositions comprising any one population of the engineered NK cells described herein. Also provided are compositions comprising any one population of the engineered T cells described herein. Also provided are compositions comprising any one population of the engineered CD34+ hematopoietic progenitor cells described herein.

[0064] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof any of the described derived cells, any of the described engineered NK cells, any of the described engineered T cells, any of the described engineered CD34+ hematopoietic progenitor cells, and any of the described compositions is disclosed.

[0065] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoma, benign lesion, bladder cancer, bone cancer, breast cancer, thyroid cancer, laryngeal carcinoma, lung carcinoma, oral carcinoma, pharyngeal carcinoma, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), cutaneous melanoma, endocrine cancer, endometrial cancer, gastrointestinal cancer, genitourinary cancer, glioblastoma, head and neck cancer, hematologic malignancy, hematopoietic cancer, Hodgkin lymphoma, intraocular melanoma, leukemia, liver cancer, lymphoma, melanoma, myeloma, myeloproliferative disorder, nervous system cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, papilloma, parathyroid cancer, prostate cancer, renal cell cancer, sarcoma, skin cancer, solid tissue carcinoma, squamous cell carcinoma, and uterine cancer.

[0066] In some aspects, a method of differentiating iPSC cells into NK cells, the method comprising subjecting any one of the described iPSC cells to a differentiation protocol that results in the production of NK cells, the differentiation protocol comprising culturing the cells in a medium comprising recombinant human IL-12 protein during the last 24 hours of culture under the differentiation protocol is provided. In some embodiments, the recombinant human IL-12 protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 33.

[0067] In one aspect, there is provided a method for differentiating iPSC cells into T cells, which comprises subjecting any one of the described iPSC cells to a differentiation protocol that includes culturing the cells in a medium containing a recombinant DLL4 variant polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 37, thereby generating T cells. A recombinant DLL4 variant polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 37 is provided.

[0068] A method for differentiating iPSC cells into CD34+ hematopoietic progenitor cells, which comprises subjecting any one of the described iPSC cells to a differentiation protocol that includes culturing the cells in a preselected medium, thereby generating CD34+ hematopoietic progenitor cells.

[0069] In one aspect, there is provided a polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-protease peptide. In many embodiments, the CD16 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the CD16 protein is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 7. In various embodiments, the CD16 protein is a CD16 variant protein. In some embodiments, the CD16 variant protein comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.

[0070] In some embodiments, the CD16 variant comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 5. In various embodiments, the CD16 variant is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 7, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In many embodiments, the NKG2D protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the NKG2D protein is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 9, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In certain embodiments, the NKG2D protein is an NKG2D variant protein. In some embodiments, the NKG2D variant comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0071] In some embodiments, the self-protease peptide is selected from the group consisting of tesehovirus-1 2A (P2A) peptide, foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, Theiler's murine encephalomyelitis virus 2A (T2A) peptide, cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and Flacherie virus 2A (BmIFV2A) peptide. In some embodiments, the self-protease peptide is a P2A peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the self-protease peptide is a P2A peptide encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 8, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0072] In some embodiments, the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein has the nucleic acid sequence of SEQ ID NO: 6.

[0073] Vectors comprising any of the described polynucleotides are provided. In some embodiments, the vector comprises, from 5' to 3', (i) a left homologous sequence, (ii) a promoter, (iii) any of the described polynucleotides, (iv) a terminator and / or polyadenylation signal sequence, and (iv) a right homologous sequence.

[0074] In some embodiments, the left homologous sequence comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 11. In some embodiments, the right homologous sequence comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 12.

[0075] In some embodiments, the vector comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 13, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 13.

[0076] In some embodiments, the vector comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 39, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 39. BRIEF DESCRIPTION OF THE DRAWINGS

[0077]

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Mode for Carrying Out the Invention

[0078] I. Introduction Provided herein are cell therapies derived from induced pluripotent stem cells (iPSCs), such as iPSC-derived natural killer (iNK) cells and iPSC-derived T (iT) cells, for immuno-oncology. In some embodiments, genetically engineered iPSC-derived immune cells express CARs and other molecules that can mediate the persistence, functionality, and / or activation of these engineered immune cells. Also provided herein are methods of generating and using such iPSC-derived immune cells.

[0079] Further described are genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD16 and recombinant NKG2D. In some embodiments, such cells also express a CAR. Also provided are related constructs (e.g., vectors), polynucleotides, and pharmaceutical compositions.

[0080] II. Definitions Unless otherwise defined, all technical and scientific terms, notations, and other technical or nomenclatural and scientific or nomenclatural terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. All patents and publications referred to herein are incorporated by reference in their entirety.

[0081] Unless otherwise defined, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Unless otherwise indicated, chemical and biochemical abbreviations and symbols are according to IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values defining the range and all integer values therebetween.

[0082] As used herein, the articles "a" and "an" refer to one, or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0083] Furthermore, as used herein, "and / or" shall be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A (alone)", as well as "B (alone)".

[0084] The term "about" as used herein is understood by those of ordinary skill in the art and varies to some extent depending on the context in which it is used. When referring to measurable values such as amounts, time periods, etc., "about" as used herein means to encompass variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, because such variations are appropriate for carrying out the disclosed method.

[0085] The terms "comprises", "comprising", "includes", "including", "has", "having", "contains", or "containing", or any other variation thereof, are understood to imply the inclusion of the 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 apparatus that includes a list of elements is not necessarily limited to those elements and can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.

[0086] As used throughout the specification and claims, the term "consists of" as used herein, or variations such as "consist of" or "consisting of", indicates that it includes any recited integer or group of integers, but cannot add additional integers or groups of integers to the specified method, structure, or composition.

[0087] As used throughout the specification and claims, the term "consists essentially of" as used herein, or variations such as "consist essentially of" or "consisting essentially of", indicates the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not substantially change the basic or novel characteristics of the specified method, structure, or composition. See M.P.E.P. § 2111.03.

[0088] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to all elements in the series. One of ordinary skill in the art will be able to understand and identify numerous equivalents to the specific embodiments of the applications described herein without performing more than routine experimentation. Such equivalents are intended to be encompassed by this application.

[0089] As used herein, "subject" means any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein includes 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 more preferably humans.

[0090] In the context of two or more nucleic acid or polypeptide sequences (e.g., CAR polypeptides and the CAR polynucleotides encoding them), the terms “identical” or percent “identity” refer to two or more sequences or subsequences that are the same, or have the same amino acid residues or nucleotides to a specified percentage, when compared and aligned for maximum correspondence, measured using one of the following sequence comparison algorithms or by visual inspection.

[0091] For sequence comparison, typically one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0092] Optimal alignment of arrays for comparison can be carried out, 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, Wisconsin), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0093] Examples of algorithms suitable for determining percent sequence identity and 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 analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued 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 serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as far as possible while increasing the cumulative alignment score.

[0094] The cumulative score is calculated using parameters M (reward score for matching residue pairs, always >0) and N (penalty score for mismatched residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of a word hit in each direction stops when: the cumulative alignment score decreases by an amount X from its maximum achieved value, the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments, or 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 initial settings, a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, as initial settings, a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0095] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see also, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides 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 similar to a reference sequence if the minimum total probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0096] A further indication 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 typically substantially identical to a second polypeptide when, for example, the two polypeptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0097] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, protein, or cell) is substantially separated from, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues. 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. An "isolated" nucleic acid, peptide, protein, and cell can still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell and is part of the composition. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0098] As used herein, the term "polynucleotide" is also synonymously referred to as "nucleic acid molecule", "nucleotide", "nucleic acid", or "polynucleic acid", and refers to any polyribonucleotide or polydeoxyribonucleotide, which can 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, hybrid molecules that can be single-stranded, or more typically double-stranded or a mixture of single-stranded and double-stranded regions, including DNA and RNA. Furthermore, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone is modified for reasons of stability or otherwise. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also often includes relatively short nucleic acid strands often referred to as oligonucleotides.

[0099] "Construct" refers to a polymer or molecular complex containing a polynucleotide that is delivered to host cells either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, which can be replicated and / or expressed in the target cell. 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, Sendai viral vectors, and the like.

[0100] "Integration" means that one or more nucleotides of a construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of a construct are inserted into a preselected site or "integration site" within the chromosomal or mitochondrial DNA of the cell. As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of the endogenous sequence or nucleotides at the integration site. If a deletion exists at the insertion site, "integration" can further include the replacement of the deleted endogenous sequence or nucleotides with one or more inserted nucleotides.

[0101] As used herein, the term "exogenous" is intended to mean that the referenced molecule or activity is introduced into or is non-native to the host cell. A molecule can be introduced, for example, by introduction of the coding nucleic acid into the host genetic material such as by integration into the host chromosome, or as extrachromosomal genetic material such as a plasmid. Thus, this term as used in reference to the expression of a coding nucleic acid refers to the introduction of the coding nucleic acid into the cell in an expressible form. 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 a coding nucleic acid, this term refers to the expression of a coding nucleic acid that is naturally contained within the cell and not exogenously introduced.

[0102] As used herein, "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed in vivo into RNA and, in some instances, translated into a polypeptide. Genes or polynucleotides of interest include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest can encode, among other things, an miRNA, shRNA, native polypeptide (i.e., a polypeptide found in nature), or a fragment thereof, a mutant polypeptide (i.e., a mutant of a native polypeptide having less than 100% sequence identity to the native polypeptide), or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0103] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that one function is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA when 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 regulatory sequences in a sense or antisense orientation.

[0104] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term encompasses the transcription of a gene into RNA. This term also encompasses the translation of the RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed CAR can be within the cytoplasm of the host cell, within the extracellular environment such as the growth medium of cell culture, or immobilized on the cell membrane.

[0105] As used herein, the terms "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by one of ordinary skill in the art. The conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. This term also encompasses amino acid polymers that are modified either naturally or by intervention, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides containing, for example, one or more analogs of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0106] The peptide sequences described in this specification are described according to the normal convention where the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Amino acid isomers are known, but the L-form of the amino acid is represented unless otherwise clearly indicated.

[0107] As used herein, the term "engineered immune cell" refers to an immune cell, also called an immune effector cell, that has been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the cell's total genetic material.

[0108] As used herein, "porcine teschovirus-1 2A peptide" or "P2A peptide" or "P2A" refers to the "self-cleaving peptide" of picornavirus. The average length of the P2A peptide is 18-22 amino acids. The P2A peptide was first identified in foot-and-mouth disease virus (FMDV), a member of the picornavirus (Ryan et al., J Gen Virol, 1991, 72(Pt 11): 2727-2732). It has been reported that ribosomes skip the synthesis of the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, resulting in cleavage between the 2A peptide and the peptide immediately downstream (see, for example, Donnelly et al., J Gen Virol., 2001, 82: 1013-1025).

[0109] As used herein, the term "differentiation" refers to the process by which unspecialized (or "uncommitted") or less specialized cells acquire the characteristics of specialized cells. Examples of specialized cells include blood cells or muscle cells. Differentiated or induced-differentiated cells have taken a more specialized (or "committed") position within the cell lineage. When applied to the process of differentiation, the term "committed" refers to a cell that, in the differentiation pathway, under normal circumstances, continues to differentiate into a specific cell type or a partial set of cell types and, under normal circumstances, cannot differentiate into different cell types or revert to a less differentiated cell type that is further along in the process. As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or the cell mass or embryo proper. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, namely, the ectoderm, mesoderm, and endoderm. Pluripotency is a continuous connection of developmental potential within the range of more primitive and more pluripotent cells that can give rise to a complete organism (such as embryonic stem cells) from less complete or partially pluripotent cells (such as epiblast stem cells or EpiSCs) that cannot give rise to a complete organism.

[0110] As used herein, the term "reprogramming" or "dedifferentiation" refers to a method of increasing the potential of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an un-reprogrammed state. In other words, a reprogrammed cell is in a less differentiated state than the same cell in an un-reprogrammed state.

[0111] As used herein, the term "induced pluripotent stem cell" or "iPSC" means a stem cell that has been induced, changed, or reprogrammed from a differentiated adult, neonatal, or fetal cell such that the stem cell can differentiate into cells of all three germ layers or cortical layers: mesoderm, endoderm, and ectoderm. The generated iPSCs do not refer to cells as found in nature.

[0112] The term "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" or "HPC" refers to cells that are committed to the hematopoietic system but are capable of further hematopoietic differentiation. Examples of hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hemocytoblasts), myeloid precursors, megakaryocyte precursors, erythroid precursors, and lymphoid precursors. 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), as well as lymphoid lineages (T cells, B cells, NK cells). As used herein, "CD34+ hematopoietic progenitor cells" refers to HPCs that express CD34 on their surface.

[0113] As used herein, the term "immune cell" or "immune effector cell" refers to a cell involved in an immune response. Examples of immune responses include, for example, promotion of immune effector responses. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytic cells.

[0114] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a type of white blood cell that matures in the thymus and has various roles in the immune system. T cells may have roles including, for example, the identification of specific foreign antigens in the body and the activation and inactivation of other immune cells. T cells can be any T cells, such as cultured T cells, for example primary T cells, or T cells from a cultured T cell line, such as Jurkat, SupTl, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cells, can be at any stage of development, and include, but are not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (gd T cells or γδ T cells), etc. Further types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can 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 can also be differentiated from stem cells or progenitor cells.

[0115] The term "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. These are characterized by their secretion profiles after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" was originally identified as a differentiation antigen on T lymphocytes but is also found on other cells including monocytes / macrophages and is a 55kD glycoprotein. The CD4 antigen is a member of the immunoglobulin supergene family and has been implicated as an associative recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes, these define the helper / inducer subset.

[0116] As used herein, the term "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 as well as on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is an associative recognition element in major histocompatibility complex class I-restricted interactions.

[0117] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of the T cell receptor (TCR chain). NK cells can also refer to genetically engineered NK cells such as NK cells modified to express a chimeric antigen receptor (CAR). NK cells can also be differentiated from stem cells or progenitor cells.

[0118] As used herein, the term "gene imprinting" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties in donor cells or iPSCs, and can be retained in iPSCs derived from donor cells and / or hematopoietic cells derived from iPSCs. As used herein, "donor cells" are non-pluripotent cells that can be used to generate iPSCs through reprogramming, and iPSCs derived from donor cells can further differentiate into specific cell types including any hematopoietic cells. iPSCs derived from donor cells, and differentiated cells derived therefrom, may be collectively referred to as "derived" or "derivative" cells depending on the context. For example, throughout this application, derived effector cells, or derived NK or "iNK" cells, or derived T or "iT" cells are cells differentiated from iPSCs as compared to their primary counterparts obtained from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, gene imprinting conferring preferential therapeutic properties is incorporated into iPSCs either through reprogramming of selected donor cells specific to the donor, disease, or treatment response, or by introducing a genetically modified modality into iPSCs using genome editing.

[0119] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to an antigen or target. Association of the extracellular domain of the CAR with the target antigen on the surface of the target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cells. The CAR can redirect the specificity of immune effector cells and initiate the proliferation, cytokine production, phagocytosis, and / or production of molecules that can mediate the cell death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner.

[0120] As used herein, the term "signal peptide" refers to the leader sequence at the amino terminus (N-terminus) of the nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequently to cell surface expression either co-translationally or post-translationally.

[0121] As used herein, the terms "extracellular antigen-binding domain", "extracellular domain", or "extracellular ligand-binding domain" refer to the part of the CAR that is located outside the cell membrane and is capable of binding to an antigen, target, or ligand.

[0122] As used herein, the term "hinge region" or "hinge domain" refers to the part of the CAR that connects two adjacent domains of the CAR protein, namely the extracellular domain and the transmembrane domain of the CAR protein.

[0123] As used herein, the term "transmembrane domain" refers to the part of the CAR that extends across the cell membrane and anchors the CAR to the cell membrane.

[0124] As used herein, the term "hinge region" or "spacer region" generally refers to any oligopeptide or polypeptide that functions to link the extracellular domain to the transmembrane domain. The hinge region can be used to provide greater flexibility and accessibility to the extracellular domain.

[0125] As used herein, the terms "intracellular signaling domain", "cytoplasmic signaling domain", or "intracellular signaling domain" refer to the part of the CAR that is located inside the cell membrane and is capable of transmitting effector signals.

[0126] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., a NK cell or a T cell) that provides a primary cytoplasmic signaling sequence that modulates primary activation of a receptor in a stimulatory fashion with respect to at least some aspects of an immune cell signaling pathway. Stimulatory molecules include two distinct classes of cytoplasmic signaling sequences, namely those that initiate antigen-dependent primary activation (referred to as "primary signaling domains") and those that act in an antigen-independent manner to provide a secondary co-stimulatory signal (referred to as "co-stimulatory signaling domains").

[0127] In certain embodiments, the extracellular domain comprises an antigen-binding domain and / or an antigen-binding fragment. The antigen-binding fragment can be, for example, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen. The antigen-binding fragments of the present application possess one or more desirable functional properties including, but not limited to, the ability to bind with high affinity to a tumor antigen, exhibit high specificity for the tumor antigen, stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing the tumor antigen, and inhibit tumor growth in subjects and animal models in which it is needed.

[0128] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins or antibody molecules that are monoclonal or polyclonal, human, humanized, chimeric, and hybrid antibodies, as well as antibody fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) according to the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, the antibodies of the present application can be of any of the five major classes or corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3, or IgG4. The antibody light chains of vertebrate species can be assigned to one of two distinct types, namely kappa and lambda, based on the amino acid sequence of their constant domain. Thus, the antibodies of the present application can contain a kappa or lambda light chain constant domain. According to certain embodiments, the antibodies of the present application include heavy chain and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, an antibody contains an antigen-binding region composed 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 domain is also alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domain is also alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0129] 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 that tumor antigen). Furthermore, an isolated antibody is substantially free of other cellular materials and / or chemicals.

[0130] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the present application can be prepared by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals such as transgenic mice or rats having a genome comprising a human heavy chain transgene and a light chain transgene.

[0131] As used herein, the term "antigen-binding fragment" refers to, for example, 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 comprising one or more CDRs, camelized single-domain antibody, minibody, nanobody, domain antibody, bivalent domain antibody, light chain variable domain (VL), variable domain of a camel antibody (V H H), or any other antibody fragment that binds an antigen but does not contain the complete antibody structure. The antigen-binding fragment can bind the same antigen to which the parent antibody or parent antibody fragment binds.

[0132] 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).

[0133] As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region or comprises only the heavy chain variable region.

[0134] 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, which is produced using any technique known in the art. This definition of human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0135] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced and the sequence homology to that of a human antibody is increased.

[0136] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and potency, while the constant regions correspond to the sequences of an antibody derived from another mammalian species (e.g., human) in order to avoid eliciting an immune response in that species.

[0137] As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence among the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence among the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or do not substantially overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetravalent antibody molecule.

[0138] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to two or fewer epitopes or two or fewer antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises half or a fragment of an antibody having binding specificity for a first epitope and half or a fragment of an antibody having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises an scFv or a fragment thereof having binding specificity for a first epitope, and an scFv or a fragment thereof having binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises V H H having binding specificity for a first epitope and V H H having binding specificity for a second epitope.

[0139] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to a tumor antigen" refers to a dissociation constant (Kd) of 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 -10An antigen-binding domain or antigen-binding fragment that binds to a tumor antigen with a KD of less than M. The term "KD" or "Kd" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka), expressed as molar concentration (M). The KD value of an antibody can be determined using methods in the art in view of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, for example, a Biacore® system, or by using biolayer interferometry technology, for example, an Octet RED96 system. The smaller the KD value of an antigen-binding domain or antigen-binding fragment, the higher the affinity of the antigen-binding domain or antigen-binding fragment for the target antigen.

[0140] Genome editing, or genomic editing, or gene editing is used interchangeably herein and is a type of genetic manipulation in which DNA is inserted, deleted, and / or substituted in the genome of a target cell. Targeted genome editing (which is interchangeable with "targeted genomic editing" or "targeted gene editing") enables insertion, deletion, and / or substitution at a preselected site in the genome. If the endogenous sequence is deleted or disrupted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down due to the deletion or disruption of the sequence. Thus, targeted editing can also be used to accurately disrupt the expression of an endogenous gene. The term "targeted integration" is used herein as well and refers to a process that includes the insertion of one or more exogenous sequences at a preselected site in the genome, with or without deletion of the endogenous sequence at the insertion site.

[0141] As used herein, the terms “cancer,” “malignant tumor,” “neoplasm,” “tumor,” and “carcinoma” are used interchangeably herein and refer to cells exhibiting relatively abnormal, unregulated, and / or autonomous growth, whereby they exhibit an abnormal growth phenotype characterized by a marked loss of control of cell proliferation. Generally, cells of interest for treatment in the present application include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings of the present disclosure may relate to any and all cancers. Non-limiting examples of one or more cancers include, for example, hematopoietic cancers such as leukemia, lymphoma (Hodgkin's and non-Hodgkin's), myeloma, and myeloproliferative disorders, sarcoma, melanoma, adenoma, solid tissue carcinomas, squamous cell carcinomas of the oral cavity, pharynx, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer, renal cell cancer, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancers, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal tract cancers and nervous system cancers, benign lesions such as papilloma, and the like.

[0142] III. NKG2D and CD16 Transgenes Methods for exogenously expressing or overexpressing CD16 and NKG2D proteins and transgenes in cells, as well as such cells and their therapeutic uses, are described herein. The surface receptor CD16 (FcγRIIIA) affects human natural killer (NK) cells during maturation. NK cells bind to the Fc portion of IgG via CD16 and perform antibody-dependent cellular cytotoxicity, which is critical for the efficacy of some anti-tumor monoclonal antibody therapies. NKG2D is a stimulatory / activating receptor that is mostly expressed on the cells of the cytotoxic arm of the immune system, including NK cells and T cell subsets. NKG2D is critical in diverse aspects of natural and acquired immune functions. In some embodiments, CD16 and NKG2D are expressed from a single polynucleotide construct because it is advantageous to reduce the number of gene edits in the cells.

[0143] In some embodiments, iPSC cells or derivatives thereof containing an exogenous or isolated polynucleotide construct encoding a CD16 protein and an NKG2D protein are provided. In some embodiments, iPSC cells or derivatives thereof that express a recombinant CD16 protein and a recombinant NKG2D protein are described herein. In some embodiments, the recombinant protein is encoded by an exogenous or isolated polynucleotide construct. In some embodiments, the polynucleotide construct encoding the CD16 protein and the NKG2D protein also includes a polynucleotide sequence encoding a self-protease peptide or a self-cleaving peptide. In some embodiments, an exogenous polynucleotide construct encoding a CD16 protein, an NKG2D protein, and a self-cleaving peptide is introduced into iPSC cells or derivatives thereof. The exogenous or isolated polynucleotide construct can be introduced into the gene locus of iPSC cells or derivatives thereof.

[0144] In some embodiments, iPSC cells or derivatives thereof that express a recombinant CD16 protein and a recombinant NKG2D protein also express a chimeric antigen receptor (CAR). In some embodiments, cells that express a recombinant CD16 protein and a recombinant NKG2D protein express either or both of recombinant HLA-E and HLA-G. In some embodiments, iPSC cells or derivatives thereof that express a recombinant CD16 protein and a recombinant NKG2D protein express a CAR and either or both of recombinant HLA-E and HLA-G. In many embodiments, cells that express a recombinant CD16 protein, a recombinant NKG2D protein, and a CAR also express a recombinant IL-15 protein. In many embodiments, the cells express a recombinant CD16 protein, a recombinant NKG2D protein, a CAR, a recombinant IL-15 protein, and either or both of recombinant HLA-E and HLA-G.

[0145] In many embodiments, cells expressing the recombinant CD16 protein, the recombinant NKG2D protein, and the CAR also express a recombinant fusion protein containing IL-15 and IL-15Rα. In many embodiments, the cells express either or both of the recombinant CD16 protein, the recombinant NKG2D protein, the CAR, the recombinant fusion protein containing IL-15 and IL-15Rα, and the recombinant HLA-E, HLA-G. In some embodiments, cells expressing the recombinant CD16 protein and the recombinant NKG2D protein also express the recombinant IL-15 protein. In some embodiments, cells expressing the recombinant CD16 protein and the recombinant NKG2D protein also express a recombinant fusion protein containing IL-15 and IL-15Rα. In some embodiments, cells expressing the recombinant CD16 protein, the recombinant NKG2D protein, and the recombinant IL-15 protein also express the CAR. In some embodiments, cells expressing the recombinant CD16 protein, the recombinant NKG2D protein, and a recombinant fusion protein containing IL-15 and IL-15Rα also express the CAR.

[0146] In one aspect, an exogenous or isolated polynucleotide construct encoding a CD16 protein and an NKG2D protein is provided. In some embodiments of the exogenous polynucleotide construct, the polynucleotide sequence encoding the CD16 protein and the polynucleotide sequence encoding the NKG2D protein are operably linked by a polynucleotide sequence encoding a self-protease peptide or a self-cleaving peptide. In some embodiments, the polynucleotide construct comprises, from the 5' to the 3' end, a polynucleotide sequence encoding a CD16 protein, a polynucleotide sequence encoding a self-protease peptide or a self-cleaving peptide, and a polynucleotide sequence encoding an NKG2D protein. In some embodiments, the polynucleotide construct comprises, from the 5' to the 3' end, a polynucleotide sequence encoding an NKG2D protein, a polynucleotide sequence encoding a self-protease peptide or a self-cleaving peptide, and a polynucleotide sequence encoding a CD16 protein. In some embodiments, the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the exogenous polynucleotide construct encodes the amino acid sequence of SEQ ID NO: 1.

[0147] In some embodiments, the CD16 protein (also referred to as "low-affinity immunoglobulin gamma Fc region receptor III-A" or "Fc gamma receptor IIIa") is a wild-type CD16 protein. In some embodiments, the human wild-type CD16 protein has the amino acid sequence set forth in NCBI Reference Sequence NP_000560.7 or UniProt number P08637. In some cases, the coding sequence of human wild-type CD16 is set forth in NCBI Reference Number NM_000569.8.

[0148] In some embodiments, the CD16 protein is a CD16 variant protein. In some examples, the CD16 variant protein has an amino acid sequence having at least 90%, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wild-type CD16 such as that of SEQ ID NO: 5. In some examples, the CD16 variant is a high-affinity CD16 variant. In other cases, the CD16 variant is a non-cleavable CD16 variant. In some examples, the CD16 variant is a high-affinity and non-cleavable CD16 variant.

[0149] In some embodiments, the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an F158V substitution, and one or more substitutions selected from F176V, S197P, D205A, S219A, T220A, and any combination thereof. In one embodiment, the CD16 variant has an F176V substitution, and one or more substitutions selected from F158V, S197P, D205A, S219A, T220A, and any combination thereof. In many embodiments, the CD16 variant has an S197P substitution, and one or more substitutions selected from F158V, F176V, D205A, S219A, T220A, and any combination thereof. In various embodiments, the CD16 variant has a D205A substitution, and one or more substitutions selected from F158V, F176V, S197P, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a substitution, and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an S219A substitution, and one or more substitutions selected from F158V, F176V, S197P, D205A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a T220A substitution, and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant protein has an F176V substitution. In some embodiments, the variant CD16 protein has the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding the variant CD16 protein has the sequence of SEQ ID NO: 7. In some embodiments, the wild-type CD16 protein has the sequence of SEQ ID NO: 5.

[0150] In some embodiments, the NKG2D protein (also known as NKG2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily K member 1, or KLRK1) is a wild-type NKG2D protein. In some embodiments, the human wild-type NKG2D protein has the amino acid sequence set forth in NCBI Reference Sequence No. NP_001186734.1 or NP_031386.2 or UniProt No. P26718. In some cases, the coding sequence of human wild-type NKG2D is set forth in NCBI Reference No. NM_001199805.1 or NM_007360.3. In some embodiments, the NKG2D protein is an NKG2D variant protein. In some examples, the NKG2D variant protein has an amino acid sequence having at least 90% sequence identity to wild-type NKG2D, such as, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, such as the one of SEQ ID NO: 4. In some embodiments, the NKG2D protein has the amino acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid sequence encoding the NKG2D protein has the sequence of SEQ ID NO: 9.

[0151] A. Self-protease peptide As described above, constructs containing self-protease peptide sequences comprising 2A peptides capable of inducing ribosome skipping during translation of polypeptides are provided herein. The 2A peptide functions to "cleave" the mRNA transcript by causing the ribosome to skip synthesis of the peptide bond between a glycine (G) and a proline (P) residue at the C-terminus, thereby effecting separation between the end of the 2A sequence and the next downstream peptide. Examples of 2A peptides include, but are not limited to, the porcine teschovirus-1 2A (P2A) peptide, the foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, the equine rhinitis A virus (ERAV) 2A (E2A) peptide, the Thosea asigna virus 2A (T2A) peptide, the cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and the flacherie virus 2A (BmIFV2A) peptide.

[0152] Exemplary P2A peptides can include amino acid sequences having at least 90% sequence identity to SEQ ID NO: 3, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the P2A peptide has the amino acid sequence of SEQ ID NO: 3.

[0153] IV. Chimeric Antigen Receptor (CAR) In some embodiments, the iPSC cells or their derivative cells contain an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR that targets a tumor antigen. In some examples, the recombinant CAR polypeptide comprises at least an extracellular domain that specifically binds to an antigen (or antigens), a transmembrane domain, and an intracellular signaling domain. In some examples, the recombinant CAR polypeptide comprises a signal peptide, an extracellular domain that specifically binds to an antigen (or antigens), a transmembrane domain, an intracellular signaling domain, and one or more co-stimulatory domains. In other instances, the recombinant CAR polypeptide comprises a signal peptide, an extracellular domain that specifically binds to an antigen (or antigens), a hinge region, a transmembrane domain, an intracellular signaling domain, and one or more co-stimulatory domains.

[0154] A. Extracellular domain In certain embodiments, the extracellular domain of the CAR comprises an antibody, an antibody fragment, an antigen-binding domain, and / or an antigen-binding fragment. The antigen-binding fragment can be, for example, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor antigen. In some embodiments, the antigen-binding domain or fragment has one or more desirable functional properties, including, but not limited to, high-affinity binding to a tumor antigen, high specificity for the tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing the tumor antigen, and the ability to inhibit tumor growth in a subject and animal model in which it is needed.

[0155] In some embodiments, antibodies or antibody fragments suitable for use in a CAR include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fv (sdFv), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals (“SMIP™”), intracellular antibodies, minibodies, single-domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the foregoing. Antibodies and / or antibody fragments can be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, variable domains of camel antibodies and humanized versions thereof, variable domains of shark antibodies and humanized versions thereof, and camelized antibody variable domains.

[0156] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, an 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 tribody, or a decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.

[0157] In some embodiments, the extracellular domain of the CAR is a single-domain antibody or a nanobody. In some embodiments, the extracellular domain is a VHH. In some embodiments, the extracellular domain is an scFv.

[0158] Optionally, alternative scaffolds for immunoglobulin domains that exhibit similar functional features such as high affinity and specific binding to target biomolecules may also be used in the described CARs. Such scaffolds have been shown to give molecules with improved features such as higher stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds include tenascin type III domains derived from engineered tenascin (e.g., Centyrin™), scaffolds derived from engineered gamma-B crystallin or scaffolds derived from engineered ubiquitin (e.g., Affilins), the tenth fibronectin type III (10Fn3) domain derived from engineered fibronectin (e.g., monobodies, Adnectins™, or AdNexins™), engineered ankyrin repeat motif-containing polypeptides (e.g., DARPins™), the A domain derived from engineered low density lipoprotein receptor (LDLR-A) (e.g., Avimers™), lipocalins (e.g., anticalins), Kunitz domains derived from engineered protease inhibitors (e.g., EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2), the Z domain derived from engineered protein A (Affibodies™), polypeptides derived from Sac7d (e.g., Nanoffitins® or affitins), the SH2 domain derived from engineered Fyn (e.g., Fynomers®), CTLD3 (e.g., tetranectin), thioredoxin (e.g., peptide aptamers), KALBITOR®, beta-sandwiches (e.g., iMab), miniproteins, C-type lectin-like domain scaffolds, engineered antibody mimetics, and any genetically engineered counterparts of the foregoing that retain their binding functionality (each of which is incorporated by reference in its entirety, 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 Biotechnol 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, Curr 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)). In some embodiments, the alternative scaffold is Affilin or Centyrin.

[0159] i. Antigen-binding domain In some embodiments, the antigen-binding domain of the CAR binds to a target antigen. The antigen-binding domain can bind to multiple antigens or multiple epitopes in one antigen. For example, the antigen-binding domain can bind to 2, 3, 4, 5, 6, 7, 8, or more antigens. As another example, the antigen-binding domain can bind to 2, 3, 4, 5, 6, 7, 8, or more epitopes in the same antigen.

[0160] The choice of antigen-binding domain can depend on the type and number of antigens that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the CAR can be genetically modified to target a desired tumor antigen by engineering the desired antigen-binding domain to specifically bind to an antigen (such as one on tumor cells). Non-limiting examples of cell surface markers that can serve as targets for the antigen-binding domain in a CAR include those associated with tumor cells or autoimmune diseases.

[0161] In some embodiments, the antigen-binding domain binds to at least one tumor antigen or autoantigen.

[0162] 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.

[0163] In some embodiments, the antigen-binding domain binds to at least one autoantigen. In some embodiments, the antigen-binding domain binds to two or more autoantigens. In some embodiments, the two or more autoantigens are associated with the same autoimmune disease. In some embodiments, the two or more autoantigens are associated with different autoimmune diseases.

[0164] 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 CD19, 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. Non-limiting examples of tumor antigens associated with renal cell carcinoma include CD70 and FOLR1.

[0165] Additional examples of antigens that can be targeted by an antigen binding domain include, but are not limited to, alpha-fetoprotein, A3, A33 antibody-specific antigen, Ba733, 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), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and 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, PAM-4 antibody-specific antigen, placental growth factor, p53, prostate acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomsen-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A-antigen, angiogenesis marker, oncogene marker, or oncogene product.

[0166] 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: 2, 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: 2 as set forth in International Application No. PCT / US / 2021 / 072646. 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: 4, 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: 4 as set forth in International Application No. PCT / US / 2021 / 072646. In one embodiment, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 7, 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: 7 as set forth in International Application No. PCT / US / 2021 / 072646.

[0167] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens can be derived from cell receptors and cells that produce antibodies directed 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 mellitus, 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-Barré syndrome, Crohn's disease, or ulcerative colitis.

[0168] In some embodiments, autoimmune antigens that can be targeted by CARs include, but are not limited to, platelet antigens, myelin protein antigens, Sm antigens in snRNPs, islet cell antigens, rheumatoid factors, and anti-citrullinated proteins, citrullinated proteins and peptides, such as CCP-1, CCP-2 (cyclic citrullinated peptides), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factors, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage, such as collagen II, IX, and XI, 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 / immunological 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 proteins, granular proteins, such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigens, islet cell antigens, rheumatoid factors, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids, such as dsDNA, ssDNA, and RNA, ribonucleoprotein particles 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.

[0169] Non-limiting exemplary antigen targets are provided in Tables 1-3. Table 1 provides antigen-binding domains that bind to exemplary antigen targets. The antigen-binding domains can include VH sequences, VL sequences, and / or their CDRs, such as those described in the cited publications, the contents of each of which are hereby incorporated by reference in their entirety for all purposes.

[0170]

Table 1-1

[0171]

Table 1-2

[0172]

Table 1-3

[0173]

Table 1-4

[0174]

Table 1-5

[0175]

Table 1-6

[0176]

Table 1-7

[0177]

Table 1-8

[0178]

Table 1-9

[0179]

Table 1-10

[0180]

Table 1-11

[0181]

Table 1-12

[0182]

Table 1-13

[0183]

Table 1-14

[0184]

Table 1-15

[0185]

Table 1-16

[0186]

Table 1-17

[0187]

Table 1-18

[0188]

Table 1-19

[0189]

Table 1-20

[0190]

Table 1-21

[0191]

Table 1-22

[0192]

Table 1-23

[0193]

Table 1-24

[0194] Table 2 provides exemplary antigen targets. The antigen-binding domain can include an scFv derived from an antibody or antibody fragment that binds to an antigen target, such as those described in the cited publications, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0195]

Table 2-1

[0196]

Table 2-2

[0197]

Table 2-3

[0198]

Table 2-4

[0199]

Table 2-5

[0200] Table 3 provides exemplary antigen targets. The antigen-binding domain can include an antigen-binding domain derived from a CAR that binds to an antigen target, such as those described in the cited publications, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0201]

Table 3-1

[0202]

Table 3-2

[0203]

Table 3-3

[0204]

Table 3-4

[0205]

Table 3-5

[0206]

Table 3-6

[0207] ii. Linker In some embodiments, the scFv fragment of the extracellular domain of the CAR comprises a linker between the VH and VL domains. The linker can be a peptide linker and can include any naturally occurring amino acids. Exemplary amino acids that can be included within the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and Phe. The linker should have a length sufficient to connect the VH and VL in such a way as to form the correct conformation relative to each other to retain the desired activity, such as binding to the antigen. The linker can be about 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 35 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15 to 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.

[0208] In some embodiments, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO: 3, 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: 3 of International Application No. PCT / US / 2021 / 072646.

[0209] In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker has the amino acid sequence set forth in SEQ ID NO: 25, or 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: 25 of International Application No. PCT / US / 2021 / 072646 and variants thereof.

[0210] Other linker sequences can include a portion of an immunoglobulin hinge region, CL, or CH1 derived from any immunoglobulin heavy or light chain isotype. Exemplary linkers that can be used include any of SEQ ID NOs: 26-56 in Table 1 of International Application No. PCT / US / 2021 / 072646, the disclosure of which including the sequence listing is incorporated herein by reference. Further linkers are described, for example, in International Publication No. WO 2019 / 060695, which is incorporated herein by reference in its entirety.

[0211] The linkers described herein, including SEQ ID NOs: 40-73 in Table 4, can be used in any of the provided polypeptides, including those containing CD16, NKG2D, IL-15, IL-15Rα, HLA-E, HLA-G, HSV-TK, PSMA, etc.

[0212] Table 4 provides exemplary linkers (SEQ ID NOs: 40-73) corresponding to SEQ ID NOs: 3 and 25-56 of U.S. Patent Application No. 17 / 657,803, filed April 4, 2022, the content of which is incorporated herein by reference in its entirety.

[0213]

Table 4

[0214] B. Signal Peptide In some embodiments, the CAR polypeptide comprises a signal peptide (e.g., a leader peptide or a localization peptide). The signal peptide can be located at the N-terminus of the extracellular domain. The signal peptide can optionally be cleaved from the extracellular domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of signal peptide sequences known to those of skill in the art can be used. Non-limiting examples of signal peptides from which the sequence can be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of various other proteins secreted by T cells. In some embodiments, the signal sequence is compatible with the secretory pathway of T cells. In certain embodiments, the signal sequence is derived from the human immunoglobulin heavy chain.

[0215] In some embodiments, the signal sequence is derived from GMCSFR. In one embodiment, the GMCSFR signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference.

[0216] C. Transmembrane Domain In some embodiments, the CAR polypeptide comprises a transmembrane domain fused in-frame between the extracellular domain and the cytoplasmic domain.

[0217] The transmembrane domain can be derived from a protein that contributes to the extracellular domain, a protein that contributes to a signaling or co-signaling domain, or a completely different protein. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution, deletion, or insertion to minimize interaction with other members of the CAR polypeptide. In some examples, the transmembrane domain is selected or modified by amino acid substitution, deletion, or insertion to avoid binding of a protein that naturally associates with the transmembrane domain. In some embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and / or an optimal distance between domains connected to the transmembrane domain.

[0218] The transmembrane domain may be derived from either a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains can be derived from the α, β, or ζ chains 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 (i.e., including at least their transmembrane regions). In some examples, the transmembrane domain may be synthetic, in which case it may predominantly contain hydrophobic residues such as leucine and valine. For example, triplets of phenylalanine, tryptophan, and / or valine can be found at each end of the synthetic transmembrane domain.

[0219] In some embodiments, it is desirable to utilize the transmembrane domain of the ζ, η, or FcεR1γ chain containing cysteine residues capable of disulfide bonding such that the resulting chimeric protein can form a disulfide-linked dimer with itself or with an unmodified version of ζ, η, or the FcεR1γ chain or related protein. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize interaction with other members of the receptor complex. In some embodiments, it is desirable to use the transmembrane domains of ζ, η, or FcεR1γ and -β, MB1 (Igα), B29, or CD3-γ, ζ, or η in order to maintain physical association with other members of the receptor complex.

[0220] In some embodiments, the transmembrane domain of the CAR is derived from CD8 or CD28. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference.

[0221] D. Hinge Region In some embodiments, the CAR polypeptide comprises a hinge region (e.g., a spacer region) between the extracellular domain and the transmembrane domain such that the extracellular domain, the hinge region, and the transmembrane domain are in-frame with each other.

[0222] The hinge region may contain up to 300 amino acids, preferably 10 - 100 amino acids, and most preferably 25 - 50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring spacer region sequence or a completely synthetic spacer region sequence. Non-limiting examples of hinge regions include part of the human CD8α chain, the partial extracellular domain of CD28, the FcγRlIIIa receptor, IgG, IgM, IgA, IgD, IgE, Ig hinge, or a functional fragment thereof. In some embodiments, additional linker 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 hinge region is derived from an immunoglobulin, the region may be mutated to prevent Fc receptor binding.

[0223] In some embodiments, the hinge region comprises the hinge domain of a recognized protein. The hinge domain may be derived from CD8α, CD28, or immunoglobulin (IgG). For example, the IgG hinge may be from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0224] In some 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 a chimera thereof. In various embodiments, the hinge domain comprises CH1, CH2, CH3, and / or the hinge region of an immunoglobulin. In many embodiments, the hinge domain comprises the core hinge region of an immunoglobulin. The term "core hinge" can be used interchangeably with the term "short hinge" ("SH"). Non-limiting examples of suitable hinge domains are the core immunoglobulin hinge regions comprising EPKSCDKTHTCPPCP from IgG1 (SEQ ID NO: 74, SEQ ID NO: 57 of International Application No. PCT / US / 2021 / 072646), ERKCCVECPPCP from IgG2 (SEQ ID NO: 75, SEQ ID NO: 58 of International Application No. PCT / US / 2021 / 072646), ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 from IgG3 (SEQ ID NO: 76, SEQ ID NO: 59 of International Application No. PCT / US / 2021 / 072646), and ESKYGPPCPSCP from IgG4 (SEQ ID NO: 77, SEQ ID NO: 60 of International Application No. PCT / US / 2021 / 072646) (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes). In many embodiments, the hinge domain is a fragment of an immunoglobulin hinge.

[0225] 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: 21, 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: 21 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference.

[0226] In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference.

[0227] In some embodiments, the transmembrane domain and / or the hinge domain is 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.

[0228] E. Cytoplasmic domain comprising a co-stimulatory domain In some aspects, the CAR polypeptide comprises a cytoplasmic domain containing at least one intracellular signaling domain. In some embodiments, the cytoplasmic domain also comprises one or more co-stimulatory signaling domains.

[0229] The cytoplasmic domain is responsible for activating at least one of the normal effector functions (e.g., specialized functions) of the host cell (e.g., T cell) in which the CAR is located. The term "effector function" refers to the specialized function of a cell. The effector function of a T cell can be, for example, cytotoxic activity or helper activity including cytokine secretion. The signaling domain can include a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Usually the entire signaling domain is present, while in many cases it is not necessary to use the entire domain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain as long as it transmits the effector function signal. The intracellular signaling domain includes any truncated portion of the signaling domain sufficient to transmit the effector function signal. Non-limiting examples of signaling domains that can be used include, for example, signaling domains derived from DAP10, DAP12, the Fc epsilon receptor I gamma chain (FCER1G), FcRβ, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, and CD79B.

[0230] In some embodiments, the cytoplasmic domain includes a CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain includes the amino acid sequence 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 to SEQ ID NO: 6 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0231] In some embodiments, the cytoplasmic domain contains one or more co-stimulatory signaling domains. In some embodiments, the one or more co-stimulatory signaling domains are derived in whole from CD28, 4-1BB, IL2Rβ, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.

[0232] In some embodiments, the co-stimulatory signaling domain is derived from 4-1BB. In one embodiment, the 4-1BB co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8, 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: 8 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0233] In some embodiments, the co-stimulatory signaling domain is derived from IL2Rβ. In one embodiment, the IL2Rβ co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9, 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: 9 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0234] In some embodiments, the co-stimulatory signaling domain is derived from CD40. In one embodiment, the CD40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10, 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: 10 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0235] In some embodiments, the co-stimulatory signaling domain is derived from OX40. In one embodiment, the OX40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11, 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: 11 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0236] In some embodiments, the co-stimulatory signaling domain is derived from CD80. In one embodiment, the CD80 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12, 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: 12 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0237] In some embodiments, the costimulatory signaling domain is derived from CD86. In one embodiment, the CD86 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13, 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: 13 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0238] In some embodiments, the costimulatory signaling domain is derived from CD27. In one embodiment, the CD27 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 14, 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: 14 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0239] In some embodiments, the costimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 15, 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: 15 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0240] In some embodiments, the co-stimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, 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: 16 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0241] In some embodiments, the co-stimulatory signaling domain is derived from DAP10. In one embodiment, the DAP10 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17, 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: 17 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0242] In some embodiments, the co-stimulatory signaling domain is derived from DAP12. In one embodiment, the DAP12 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18, 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: 18 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0243] In some embodiments, the costimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 19, 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: 19 of International Application No. PCT / US / 2021 / 072646, the disclosure of which is hereby incorporated by reference in its entirety.

[0244] In some embodiments, the CAR polypeptide comprises one costimulatory signaling domain. In many embodiments, the CAR comprises two or more costimulatory signaling domains. In various embodiments, the CAR comprises two, three, four, five, six, or more costimulatory signaling domains.

[0245] V. Non-classical HLA class I In some embodiments, the iPSC or its derivative cells can be further modified by introducing an exogenous polynucleotide encoding one or more proteins associated with immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). In some cases, disruption of the beta-2-microglobulin (B2M) gene eliminates surface expression of all MHC class I molecules, rendering the cells vulnerable to lysis by NK cells through a "self-loss" response. Expression of exogenous HLA-E may confer resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol., 2017 Aug; 35(8): 765-772). In some embodiments, the iPSC or its derivative cells are engineered to exogenously express HLA-E and / or HLA-G. In certain embodiments, the iPSC or its derivative cells having disruption or elimination of B2M expression are engineered to exogenously express HLA-E and / or HLA-G.

[0246] In some embodiments, the iPSC or its derivative cells comprise an exogenous polypeptide encoding at least one of human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G). In some embodiments, the iPSC or its derivative cells comprise an exogenous polynucleotide encoding HLA-E, HLA-G, or both HLA-E and HLA-G. In many embodiments, the exogenous polynucleotide encodes HLA-E and HLA-G such that HLA-E and HLA-G are operably linked by a self-protease peptide. For example, the polynucleotide can comprise, in order from 5’ to 3’, an HLA-E sequence, a P2A sequence, and an HLA-G sequence. In some cases, the polynucleotide can comprise, in order from 5’ to 3’, an HLA-G sequence, a P2A sequence, and an HLA-E sequence.

[0247] In some embodiments, the full-length HLA-E protein has the amino acid sequence set forth in NCBI reference sequence NP_005507.3 or UniProt number P13747. In some cases, the coding sequence of full-length HLA-E is set forth in NCBI reference number NM_005516.5. In some embodiments, the mature HLA-E protein has the sequence from amino acid positions 22-358 of the sequence set forth in NCBI reference sequence NP_005507.3 or UniProt number P13747.

[0248] In some embodiments, the HLA-E protein comprises an amino acid sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65, preferably SEQ ID NO: 65 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference. In some embodiments, the HLA-E protein comprises an amino acid sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14, preferably SEQ ID NO: 14 as described in FIG. 5.

[0249] In some embodiments, the full-length HLA-G protein has the amino acid sequence described in NCBI Reference Sequence No. NP_002118.1 or UniProt number P17693. In some cases, the coding sequence of full-length HLA-G is described in NCBI Reference Number NM_002127.5. In some embodiments, the mature HLA-G protein has the sequence from amino acid positions 25 to 358 of the sequence described in NCBI Reference Sequence No. NP_002118.1 or UniProt number P17693.

[0250] In some embodiments, the HLA-G protein comprises an amino acid sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 68, preferably SEQ ID NO: 68 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference. In some embodiments, the HLA-G protein comprises an amino acid sequence having at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15, preferably SEQ ID NO: 15 as described in FIG. 5.

[0251] In various embodiments, the exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-E via a linker (including those described herein). In some embodiments, the exogenous polypeptide comprises an amino acid sequence having at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 66, preferably SEQ ID NO: 66 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference. In various embodiments, the exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein fused to HLA-G via a linker (including those described herein). In some embodiments, the exogenous polypeptide comprises an amino acid sequence having at least 90%, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 69, preferably SEQ ID NO: 69 as described in International Application No. PCT / US / 2021 / 072646, the disclosure of which is incorporated herein by reference.

[0252] In some examples, the exogenous polynucleotide encodes an HLA polypeptide comprising an HLA sequence such as a mature B2M protein and / or a signal peptide such as an HLA-E signal peptide linked to a mature HLA-E protein. In some embodiments, B2M and HLA-E are linked by a flexible linker such as, but not limited to, a glycine-serine linker and others described herein. In some examples, an HLA polypeptide comprising an HLA sequence such as a mature B2M protein and / or a signal peptide such as an HLA-G signal peptide linked to a mature HLA-G protein. In some embodiments, B2M and HLA-G are linked by a flexible linker such as, but not limited to, a glycine-serine linker and others described herein.

[0253] VI. IL-15 and IL-15Ra Polypeptides In some embodiments, the iPSC or its derivative cells are modified to express an exogenous polynucleotide encoding the IL-15 protein. In some embodiments, the iPSC or its derivative cells are modified to express an exogenous polynucleotide encoding a fusion protein comprising the IL-15 protein as well as interleukin-15 receptor alpha (IL-15Rα, IL-15Ra, and IL-15RA). Such exogenous polynucleotide constructs can be introduced into specific genomic sites or loci of the iPSC or derivative cells.

[0254] In some embodiments, the full-length IL-15 protein has the amino acid sequence set forth in NCBI Reference Sequence No. NP_000576.1 or NP_751915.1 or UniProt No. P40933. In some cases, the coding sequence of full-length IL-15 is set forth in NCBI Reference No. NM_000585.4 or NM_172175.2. In some embodiments, the mature IL-15 protein has the sequence from amino acid positions 49-162 of the sequence set forth in NCBI Reference Sequence No. NP_000576.1 or NP_751915.1 or UniProt No. P40933. In some embodiments, the IL-15 protein comprises an amino acid sequence having at least 90% sequence identity, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 16, preferably SEQ ID NO: 16 as set forth in FIG. 5.

[0255] In some embodiments, the full-length IL-15Ra protein has the amino acid sequence set forth in NCBI reference sequence numbers NP_001230468.1, NP_001243694.1, NP_002180.1, or NP_751950.2, or UniProt number Q13261. In some cases, the coding sequence of full-length IL-15Ra is set forth in NCBI reference numbers NM_001243539.1, NM_001256765.1, NM_002189.3, or NM_172200.2. In some embodiments, the IL-15Ra protein comprises an amino acid sequence having at least 90% sequence identity, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 18, preferably SEQ ID NO: 18 as set forth in FIG. 5.

[0256] In some embodiments, the fusion protein comprising an IL-15 protein and an interleukin-15 receptor alpha (IL-15Rα) protein comprises an amino acid sequence having at least 90% sequence identity, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 17, preferably SEQ ID NO: 17 as set forth in FIG. 5.

[0257] An exogenous polynucleotide encoding an IL-15 protein or a fusion protein comprising an IL-15 protein and an interleukin-15 receptor alpha (IL-15Rα) can be integrated into a genomic site by genome editing.

[0258] VII. Combinatorial artificial cell death / reporter system polypeptides In some embodiments, the iPSCs or their derivative cells described herein are modified to express an exogenous combined artificial cell death / reporter system polypeptide. In some embodiments, iPSCs or their derivative cells that express an exogenous combined artificial cell death / reporter system polynucleotide are described herein. In some embodiments, polynucleotides encoding a combined artificial cell death / reporter polypeptide and iPSCs or their derivatives engineered to effect expression are provided herein. In some embodiments, a combined artificial cell death / reporter polypeptide and iPSCs or their derivatives engineered to effect expression are provided herein.

[0259] The combined artificial cell death / reporter polypeptide acts as a safety switch so that cells can be killed if the patient has an adverse reaction. In some embodiments, the polypeptide or its components are useful for imaging, including but not limited to molecular imaging and PET imaging. It is advantageous to engineer the cells to include a safety switch to eliminate cells injected into the patient in the event of an adverse event.

[0260] In some embodiments, a combined artificial cell death / reporter polypeptide is provided that can function as an artificial cell death polypeptide, a reporter polypeptide, or both an artificial cell death polypeptide and a reporter polypeptide. Having the combined artificial cell death and reporter polypeptides in a single polynucleotide that can be expressed as a single polypeptide has the advantage of reducing the number of gene edits in the cell. A description of the combined artificial cell death / reporter polypeptide can be found, for example, in U.S. Patent Application Publication No. 2022 / 0332782, the contents of which are incorporated herein by reference in their entirety.

[0261] In some embodiments, the artificial cell death / reporter polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 25. Also provided is a polynucleotide encoding an artificial cell death / reporter polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 25.

[0262] In some embodiments, the artificial cell death / reporter polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 27, 30, and 31. Also provided is a polynucleotide encoding an artificial cell death / reporter polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 31.

[0263] In some embodiments, the artificial cell death / reporter polypeptide comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 26, 28, and 32. Also provided is a polynucleotide encoding an artificial cell death / reporter polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 32.

[0264] In some embodiments, the combined artificial cell death / reporter polypeptide comprises an HSV-TK fused via a linker to a truncated mutant PSMA polypeptide. In some embodiments, the combined artificial cell death / reporter polypeptide comprises (1) an intracellular domain having a herpes simplex virus thymidine kinase (HSV-TK) and a linker, (2) a transmembrane region, and (3) an extracellular domain comprising a prostate-specific membrane antigen (PSMA) extracellular domain or a fragment thereof. In some embodiments, the linker comprises a Whitlow linker, a self-cleaving protease peptide sequence, such as porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), foot-and-mouth disease virus 2A (F2A), and a self-cleaving protease peptide sequence selected from the group consisting of any of the linkers described. In some embodiments, the artificial cell death / reporter polypeptide comprises an intracellular domain of HSV-TK fused via a linker to a truncated mutant PSMA polypeptide. Thus, the PSMA portion is extracellular and the HSV-TK is intracellular. In some embodiments, there is provided a polynucleotide encoding the combined artificial cell death / reporter polypeptide and an iPSC or a derivative thereof engineered to effect expression.

[0265] In some embodiments, the artificial cell death polypeptide comprises a viral enzyme recognized by an antiviral agent. In some embodiments, the viral enzyme is herpes simplex virus thymidine kinase (HSV-TK) (see, e.g., Bonini et al., Science, 1997 Jun 13;276(5319):1719-24). In some embodiments, the HSV-TK polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23 or 29. Also provided are polynucleotides encoding an HSV-TK polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 23 or 29.

[0266] In some embodiments, the artificial cell death / reporter system polypeptide is encoded by the HSV-TK-T2A-PSMA transgene. In some embodiments, this is encoded by the HSV-TK(H168A)-T2A-PSMA transgene. In some embodiments, this is encoded by the HSV-TK(A168H)-T2A-PSMA transgene.

[0267] In some embodiments, such cells are engineered to contain a gene (a "suicide gene") for an artificial cell death polypeptide, a molecule encoded by a gene that enables selective destruction of the cell (e.g., enables selective ablation of genetically modified cells), thereby preventing collateral damage to neighboring cells and / or tissues. Artificial cell death polypeptides include engineered proteins designed to prevent potential toxicity or other deleterious effects of cell therapy. Artificial cell death / reporter polypeptides can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some embodiments, artificial cell death polypeptides are provided herein. In some embodiments, polynucleotides encoding iPSCs or derivatives thereof engineered to effect artificial cell death and expression are provided herein. In some cases, the artificial cell death polypeptide is activated by an exogenous molecule, e.g., an antibody, an antiviral agent, or a radioisotope conjugate agent, which, when activated, initiates apoptosis and / or cell death of the therapeutic cells.

[0268] A reporter polypeptide refers to, and includes, an engineered protein that can be used, in combination with an imaging probe, to label cells. In some embodiments, the reporter polypeptide includes an antigen targeted by an entity such as a small molecule compound, a radioisotope conjugate, or an antibody or antigen-binding fragment thereof. In certain embodiments, the antigen is a prostate-specific membrane antigen (PSMA) polypeptide, also called glutamate carboxypeptidase 2. PSMA is a type II membrane protein targeted to the secretory pathway by its transmembrane domain and is biochemically similar to a signal sequence without being cleaved. In various embodiments, the reporter polypeptide includes the extracellular domain of prostate-specific membrane antigen (PSMA) or a fragment thereof.

[0269] In some embodiments, the PSMA polypeptide is a truncated variant as described in International Publication No. WO 2015 / 143029 and International Publication No. WO 2018 / 187791, the disclosures of which are incorporated herein by reference in their entireties. In many embodiments, the prostate-specific membrane antigen (PSMA) polypeptide comprises, or consists of, an amino acid sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24, preferably the amino acid sequence of SEQ ID NO: 24. In many embodiments, when cells are contacted with a radioisotope conjugate drug that binds to PSMA via a peptide, the expression of truncated PSMA in the cells induces cell death of the engineered cells, so the PSMA antigen can also function as an artificial cell death polypeptide. PSMA-targeted compounds are described in International Publication No. WO 2010 / 108125, the disclosure of which is incorporated herein by reference. In some embodiments, the truncated variant PSMA polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24. In some embodiments, polynucleotides encoding truncated variant PSMA polypeptides are described that comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24.

[0270] In some embodiments, the artificial cell death / reporter polypeptide comprises a viral enzyme recognized by an antiviral agent. In certain embodiments, the viral enzyme is herpes simplex virus thymidine kinase (HSV-TK). In certain embodiments, HSV-TK comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 23, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, preferably the amino acid sequence of SEQ ID NO: 23. This enzyme phosphorylates the non-toxic prodrugs acyclovir or ganciclovir, which is then phosphorylated by endogenous kinases to GCV-triphosphate, causing chain termination and single-strand breaks upon incorporation into DNA, thereby killing dividing cells. In some embodiments, a polynucleotide encoding an HSV-TK polynucleotide encoding an amino acid sequence that is at least 90% identical to SEQ ID NO: 23, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, is described.

[0271] In some embodiments, viral enzyme expression in engineered immune cells expressing a chimeric antigen receptor (CAR) induces cell death of the engineered immune cells when the cells are contacted with one or more antiviral agents. In certain embodiments, the one or more antiviral agents comprise acyclovir or a derivative thereof, or ganciclovir or a derivative thereof.

[0272] In some embodiments, cells expressing the artificial cell death / reporter system also express one or more of the other exogenous polypeptides described. In some examples, the cells express a CAR. In some examples, the cells express a CD16 polypeptide, such as a CD16 variant. In some examples, the cells express an NKG2D polypeptide.

[0273] VIII. Other Exogenous Polypeptides In some embodiments, genome editing at one or more selected genomic sites described may include, but is not limited to, the insertion of one or more exogenous polynucleotides encoding any of a polypeptide that promotes engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of an engineered iPSC or its derivative cells, such as an artificial cell death polypeptide, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein.

[0274] Other exogenous polynucleotides encoding polypeptides may include those encoding a PET reporter, a constitutive cytokine, inhibitory checkpoint inhibitor proteins such as PD1, PD-L1, and CTLA4, and proteins targeting the CD47 / Signal Regulatory Protein Alpha (SIRPα) axis. In some embodiments, engineered iPSCs generated using the methods provided herein contain modifications by insertion or deletion (in / del) at one or more endogenous genes related to a targeting modality, a receptor, a signaling molecule, a transcription factor, a drug target candidate, regulation and modulation of the immune response, or a protein that suppresses engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of an iPSC or its derivative cells.

[0275] In some embodiments, when an engineered iPSC contains two or more suicide genes, the engineered iPSC generated using the provided methods, such that the suicide genes are integrated into different safe harbor loci such as, but not limited to, the AAVS1 locus, the CCR5 locus, the ROSA26 locus, the collagen locus, the HTRP locus, the beta-2 microglobulin locus, the GAPDH locus, the TCR locus, and the RUNX1 locus, contains one or more different exogenous polynucleotides encoding a protein including caspase, thymidine kinase, cytosine deaminase, CD20, ErbB2, or CD79b.

[0276] IX. Gene Locus In one aspect, any of the described exogenous polynucleotides can be incorporated into a specific gene locus selected from the group consisting of: AAVS1 locus, B2M locus, CIITA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPBP locus, NLRC5 locus, RFXANK locus, RFX5 locus, RFXAP locus, CISH locus, CBLB locus, SOCS2 locus, PD1 locus, CTLA4 locus, LAG3 locus, TIM3 locus, and TIGIT locus. In some embodiments, any of the described exogenous polynucleotide constructs can be incorporated into a specific gene locus selected from the group consisting of: AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, TAP1 locus, TAP2 locus, TAPBP locus, TRAC locus, TRBC1 locus, RFXANK locus, RFX5 locus, and RFXAP locus, and optionally, the incorporation into the gene locus disrupts (such as reducing or eliminating) gene expression. In some embodiments, any of the exogenous polynucleotides is incorporated into a gene locus selected from the group consisting of: AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, and TRAC locus, and optionally, the incorporation into the gene locus disrupts (such as reducing or eliminating) gene expression.

[0277] In other words, the integration of the described exogenous polynucleotide may result in the disruption of one or more genes selected from the group consisting of the AAVS1 gene, B2M gene, CIITA gene, CD70 gene, CLYBL gene, NKG2A gene, NKG2D gene, TAP1 gene, TAP2 gene, TAPBP gene, TRAC (TRA) gene, TRBC1 (TRB) gene, RFXANK gene, RFX5 gene, RFXAP gene, and any combination thereof. In some examples, the disruption is to the B2M gene. In some examples, the disruption is to the CIITA gene. In some examples, the disruption is to the B2M gene and the CIITA gene.

[0278] In some embodiments, the exogenous polynucleotide encoding the CD16 protein or variant thereof and the NKG2D protein or variant thereof is integrated within any of the described gene loci. In some embodiments, the exogenous polynucleotide encoding HLA-E is integrated within any of the described gene loci. In some embodiments, the exogenous polynucleotide encoding HLA-G is integrated within any of the described gene loci. In some embodiments, the exogenous polynucleotide encoding IL-15 is integrated within any of the described gene loci. In some embodiments, the exogenous polynucleotide encoding IL-15Ra is integrated within any of the described gene loci. In some embodiments, the exogenous polynucleotide encoding a fusion protein containing IL-15 and IL-15Ra is integrated within any of the described gene loci.

[0279] X. Targeted genome editing at selected gene loci In some embodiments, one or more of the described exogenous polynucleotides are integrated at one or more loci on the chromosome of a cell such as an iPSC. In some examples, integration of the exogenous polynucleotide within the gene locus is effected by targeted genome editing. Non-limiting examples of targeted genome editing include any method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variants thereof.

[0280] Targeted editing can be accomplished either through nuclease-independent or nuclease-dependent techniques. In nuclease-independent targeted editing techniques, homologous recombination is directed through the enzymatic machinery of the host cell by homologous sequences flanking the exogenous polynucleotide to be inserted.

[0281] Alternatively, targeted editing can be accomplished at a higher frequency through the specific introduction of double-strand breaks (DSBs) by a site-specific rare-cutting endonuclease. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), that occur in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertions or deletions (in / dels) of a few endogenous nucleotides. In comparison, 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 by homologous recombination during homologous recombination repair (HDR), resulting in "targeted integration".

[0282] Available endonucleases that can introduce specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat) systems. Additionally, the DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration.

[0283] ZFN is a targeted nuclease that includes a nuclease fused to a zinc finger DNA binding domain. The "zinc finger DNA binding domain" or "ZFBD" means a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within the zinc finger binding domain, and its structure is stabilized through coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a non-naturally occurring domain, the design / composition of which results primarily from the application of rational criteria, such as substitution rules and computerized algorithms, to process information in a database that preserves information on existing ZFP designs and binding data. See, for example, U.S. Patent No. 6,140,081, U.S. Patent No. 6,453,242, and U.S. Patent No. 6,534,261. See also International Publication No. 98 / 53058, International Publication No. 98 / 53059, International Publication No. 98 / 53060, International Publication No. 02 / 016536, and International Publication No. 03 / 016496. A "selected" zinc finger domain is a non-naturally occurring domain, the generation of which results primarily from empirical processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in further detail in U.S. Patent No. 7,888,121 and U.S. Patent No. 7,972,854, the entire disclosures of which are incorporated herein by reference. The most recognized example of ZFNs in the art is the fusion of the FokI nuclease to a zinc finger DNA binding domain.

[0284] TALEN is a targeted nuclease that includes a nuclease fused to a TAL effector DNA binding domain. The "transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" means the polypeptide domain of the TAL effector protein that mediates the binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA binding domain depends on an imperfect 34-amino acid repeat effector-variable number that includes polymorphisms at selected repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of TALENs in the art is a fusion polypeptide of the Fokl nuclease and the TAL effector DNA binding domain.

[0285] Another example of a targeted nuclease found to be useful in the subject methods is a targeted Spoll nuclease, which is a polypeptide that includes a Spoll polypeptide having nuclease activity fused to a DNA binding domain that is specific for a target DNA sequence, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, and the like. See, for example, U.S. Patent Application Publication No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0286] Further examples of targeted nucleases suitable for the present application include, but are not limited to, Bxbl, phiC3 l, R4, PhiBTl, and Wp / SPBc / TP90l-l, whether used individually or in combination.

[0287] Other non-limiting examples of engineered nucleases include naturally occurring and recombinant nucleases, CRISPR-associated nucleases from the families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr, restriction endonucleases, meganucleases, homing endonucleases, etc. As an example, CRISPR / Cas9 requires two main components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex that is recruited to a target DNA sequence containing the PAM and the seed region near the PAM. The crRNA and tracrRNA combine to form a chimeric guide RNA (gRNA) that can direct Cas9 to target the selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction. As another example, CRISPR / Cpf1 includes two main components: (1) the Cpf1 endonuclease and (2) the crRNA. When co-expressed, the two components form a ribonucleoprotein (RNP) complex that is recruited to a target DNA sequence containing the PAM and the seed region near the PAM. The crRNA combines to form a chimeric guide RNA (gRNA) that can direct Cpf1 to target the selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction.

[0288] MAD7 is an engineered Cas12a variant of bacterial origin from Eubacterium rectale, which has a preference for 5’-TTTN-3’ and 5’-CTTN-3’ PAM sites and does not require tracrRNA. See, for example, International Publication No. WO 2018 / 236548, the disclosure of which is incorporated herein by reference. Further description of the CRISPR-MAD7 method can be found, for example, in CRISPR J., April 2020, 3(2):97-108.

[0289] DICE-mediated insertion provides for the unidirectional integration of exogenous DNA that is tightly restricted to the small attB and attP recognition sites of each enzyme using a pair of recombinases, such as phiC31 and Bxbl, respectively. Since these target att sites do not naturally occur in the mammalian genome, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference, and Farriggo et al., Methods Mol Biol, 2017, 1642:69-85.

[0290] In one aspect, provided herein are constructs comprising one or more exogenous polynucleotides for targeted genomic integration. In some embodiments, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to enable site-specific homologous recombination by the enzymatic machinery of the cell host. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site to enable ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site to enable TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a gRNA comprising a Cpf1 expression cassette and a guide sequence specific to a desired integration site to enable Cpf1-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and a gRNA comprising a Cas9 expression cassette and a guide sequence specific to a desired integration site to enable Cas9-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing into a desired integration site in the cell a construct comprising one or more "att" sites of a pair of DICE recombinases, introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing an expression cassette for the DICE recombinase to enable DICE-mediated targeted integration.

[0291] Genomic safe harbors that can serve as sites for targeted integration include, but are not limited to, intragenic or extragenic regions of the human genome that can accommodate predictable expression of newly integrated DNA without a deleterious effect on the host cell or organism. In certain embodiments, the genomic safe harbor for targeted integration is one or more loci of a gene selected from the group consisting of the AAVS1, CCR5, ROSA26, HTRP, GAPDH, TCR, and RUNX1 genes. In some embodiments, the TCR gene is selected from the group consisting of the TRA gene, the TRB gene, the TRD gene, and the TRG gene.

[0292] In other embodiments, the site for targeted integration is selected for deletion or reduced expression of the endogenous gene at the insertion site. Deletions related to gene expression include any genetic modification that abolishes gene expression. Examples of deletions in gene expression include, for example, removal or deletion of the DNA sequence of the gene that abolishes gene expression, insertion of an exogenous polynucleotide sequence at the locus of the gene, and one or more substitutions within the gene.

[0293] Genes for target deletion include, but are not limited to, genes for major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must match for histocompatibility in an allogeneic recipient in order to avoid the problem of allogeneic rejection. MHC deficiency, including MHC-class I deficiency, or MHC-class II deficiency, or both, refers to cells that either lack, no longer maintain, or have a reduced level of surface expression of the complete MHC complex containing the MHC class I protein heterodimer and / or the MHC class II heterodimer, where the attenuated or reduced level is lower than the level naturally detectable by other cells or by synthetic methods. MHC-class I deficiency can be achieved by a functional deletion of any region of the MHC-class I locus (chromosome 6p21), or by deleting or reducing the expression level of one or more MHC-class I-related genes including, but not limited to, the beta-2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 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. MHC-class II deficiency can be achieved by a functional deletion or reduction of MHC-II-related genes including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through activation of the transcription factor RFX5 required for the expression of class II proteins. CIITA null cells are MHC-II deficient. In some embodiments, one or more of the exogenous polynucleotides are integrated into one or more loci of a gene selected from the group consisting of the B2M, TAP1, TAP2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes, thereby deleting or reducing the expression of the gene upon integration.

[0294] In some embodiments, the exogenous polynucleotide is integrated into one or more loci on the chromosome of the cell, preferably, one or more loci are loci of genes selected from the group consisting of AAVS1, CCR5, ROSA26, HTRP, GAPDH, RUNX1, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRa constant region, TCRb constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided that at least one of the one or more loci is a locus of a gene selected from the group consisting of MHC genes, for example, genes selected from the group consisting of B2M, TAP1, TAP2, Tapasin, RFXANK, CIITA, RFX5, and RFXAP genes. In many embodiments, one or more exogenous polynucleotides are integrated into loci of MHC class I-related genes, for example, the B2M gene, the TAP1 gene, the TAP2 gene, or the Tapasin gene, and loci of MHC-II-related genes, for example, RFXANK, CIITA, RFX5, RFXAP, or CIITA genes, and optionally further into loci of safe harbor genes selected from the group consisting of AAVS1, CCR5, ROSA26, HTRP, GAPDH, TCR, and RUNX1 genes. In some embodiments, the TCR gene is selected from the group consisting of TRA gene, TRB gene, TRD gene, and TRG gene. In various embodiments, one or more of the exogenous polynucleotides are integrated into loci of CIITA, AAVS1, and B2M genes.

[0295] In some embodiments, (i) the exogenous polynucleotide is integrated into a genetic locus, (ii) a different exogenous polynucleotide is integrated into the locus of the CIITA gene, (iii) another different exogenous polynucleotide is integrated into the locus of the B2M gene, and the integration of the exogenous polynucleotide results in deletion or reduction of the expression of the CIITA and B2M genes. In some embodiments, (i) the exogenous polynucleotide is integrated into a genetic locus, (ii) another exogenous polynucleotide is integrated into the locus of the CIITA gene, (iii) yet another exogenous polynucleotide is integrated into the locus of the B2M gene, and the integration of the exogenous polynucleotide eliminates or reduces the expression of the CIITA and B2M genes. In certain embodiments, (i) the first exogenous polynucleotide is integrated into a safe harbor locus, (ii) the second exogenous polynucleotide is integrated into the locus of the CIITA gene, (iii) the third exogenous polynucleotide is integrated into the locus of the B2M gene, and the integration of the exogenous polynucleotide eliminates or reduces the expression of the CIITA and B2M genes. In some embodiments, (i) the first exogenous polynucleotide is integrated into the AAVS1, CCR5, ROSA26, HTRP, GAPDH, TRA, TRB, TRD, TRG, or RUNX1 gene locus, (ii) the second exogenous polynucleotide is integrated into the CIITA gene locus, (iii) the third exogenous polynucleotide is integrated into the B2M gene locus, and the integration of the exogenous polynucleotide eliminates or reduces the expression of the CIITA and B2M genes.

[0296] In some embodiments, the exogenous polynucleotide is integrated into the CD70 gene locus. In some embodiments, (i) the exogenous polynucleotide is integrated into a genetic locus, (ii) a different exogenous polynucleotide is integrated into the locus of the CIITA gene, (iii) another different exogenous polynucleotide is integrated into the locus of the B2M gene, (iv) yet another different exogenous polynucleotide is integrated into the CD70 gene locus, and the integration of the exogenous polynucleotide eliminates or reduces the expression of the CD70, CIITA, and B2M genes.

[0297] In some embodiments, (i) the first exogenous polynucleotide is integrated into a genetic locus, (ii) the second exogenous polynucleotide is integrated into the CIITA genetic locus, (iii) the third exogenous polynucleotide is integrated into the B2M genetic locus, (iv) the fourth exogenous polynucleotide is integrated into the CD70 genetic locus, and the integration of the exogenous polynucleotides eliminates or reduces the expression of the CD70, CIITA, and B2M genes.

[0298] In certain embodiments, (i) the first exogenous polynucleotide is integrated into a safe harbor genetic locus, (ii) the second exogenous polynucleotide is integrated into the CIITA genetic locus, (iii) the third exogenous polynucleotide is integrated into the B2M genetic locus, (iv) the fourth exogenous polynucleotide is integrated into the CD70 genetic locus, and the integration of the exogenous polynucleotides eliminates or reduces the expression of the CD70, CIITA, and B2M genes.

[0299] In some embodiments, (i) the first exogenous polynucleotide is integrated into the AAVS1, CCR5, ROSA26, HTRP, GAPDH, TRA, TRB, TRD, TRG, or RUNX1 genetic locus, (ii) the second exogenous polynucleotide is integrated into the CIITA genetic locus, (iii) the third exogenous polynucleotide is integrated into the B2M genetic locus, (iv) the fourth exogenous polynucleotide is integrated into the CD70 genetic locus, and the integration of the exogenous polynucleotides eliminates or reduces the expression of the CD70, CIITA, and B2M genes.

[0300] XI. Derived cells from XI.iPSC cells In some embodiments, cells differentiated from iPSC cells or their derivatives are provided. In some embodiments, iPSCs are differentiated into a cell type and then cultured and differentiated into another cell type. For example, iPSCs can be differentiated into progenitor cells such as NK progenitor cells and then cultured under conditions to become mature cells such as NK cells. As described above, genome editing introduced into iPSCs is retained in the derived cells. In some embodiments of the derived cells obtained from iPSC differentiation, the derived cells include, but are not limited to, hematopoietic stem and progenitor cells (HSCs), hematopoietic multipotent progenitor cells, T cell precursors, natural killer (NK) cell precursors, B cell precursors, CD34+ hematopoietic progenitor cells, T cells, NKT cells, NK cells, B cells, antigen-presenting cells (APCs), monocytes, and macrophages. In some embodiments, the derived cells are immune effector cells such as NK cells or T cells.

[0301] In some embodiments, iPSCs are generated from whole peripheral blood mononuclear cells. In some embodiments, iPSCs are generated from NK cells. In some embodiments, iPSCs are generated from T cells. In some embodiments, iPSCs are generated from reprogrammed NK cells. In some embodiments, iPSCs are generated from reprogrammed T cells. In some embodiments, iPSCs are generated from reprogrammed whole peripheral blood mononuclear cells.

[0302] Also provided is a method for producing differentiated cells or their derivatives. In some embodiments, it includes differentiating iPSCs under conditions to promote, facilitate, or generate specific differentiated cells. In some examples, the differentiated cells are further cultured to generate cells derived from the differentiated cells, such as derived cells.

[0303] iPSCs can be differentiated by any method known in the art. Exemplary methods are described in U.S. Patent No. 10,947,502, U.S. Patent No. 8,846,395, U.S. Patent No. 8,945,922, U.S. Patent No. 8,318,491, International Publication No. 2010 / 099539, International Publication No. 2010 / 141801, International Publication No. 2012 / 109208, International Publication No. 2016 / 010148, International Publication No. 2017 / 070333, International Publication No. 2017 / 070337, International Publication No. 2017 / 179720, International Publication No. 2018 / 048828, International Publication No. 2019 / 157597, and International Publication No. 2020 / 252477, the contents of which are incorporated herein by reference in their entirety. Differentiation protocols can use feeder cells or be feeder-free. A feeder cell or feeder is a type of cell that, when co-cultured with a second type of cell, provides an environment in which the second type of cell can grow, expand, or differentiate because the feeder cell provides stimuli, growth factors, and nutrients for the support of the second cell type.

[0304] In one embodiment, the differentiated iPSCs are NK cells prepared by a method of differentiating iPSCs into NK cells. In some embodiments, the iPSCs are subjected to a differentiation protocol that includes adding recombinant human IL-12 p70 (e.g., IL-12) to the culture medium during the last 24 hours of culture. By including IL-12 in the differentiation protocol, cells primed with IL-12 exhibit more rapid cell killing compared to those differentiated in the absence of IL-12. Furthermore, cells differentiated using the IL-12 condition exhibit improved cancer cell growth inhibition. Descriptions of methods for generating iPSC-derived NK cells can be found, for example, in International Publication No. WO 2010 / 099539, Euchner et al., Front Immunol, 2021 May 04, 12:640672, Li et al., Cell Stem Cell, 2018 Aug 2, 23:181-192, and Karagiannis and Kim, Mol Cells, 2021 Aug 31, 44(8):541-548. In many embodiments, recombinant human IL-12 p70 (human IL-12) comprises an IL-12 p40 subunit and / or an IL-12 p35 subunit. In many embodiments, the IL-12 p40 subunit is connected to the IL-12 p35 subunit by a linker that can be any of those described herein. In some embodiments, recombinant human IL-12 p70 comprises an IL-12 p40 subunit, a Whitlow linker, and an IL-12 p35 subunit. In some embodiments, the recombinant human IL-12 p70 protein has at least 90% sequence identity to SEQ ID NO: 33, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the recombinant human IL-12 p70 protein is encoded by a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34.

[0305] In many embodiments, the differentiated iPSCs are T cells prepared by a method of differentiating iPSCs into T cells. Protocols for generating T cells from iPSCs include, for example, those disclosed in International Publication Nos. 2010 / 099539, 2010 / 141801, 2017 / 070333, 2017 / 070337, 2017 / 179720, 2018 / 048828, 2019 / 157597, and 2020 / 252477, the contents of which are incorporated herein by reference in their entirety.

[0306] In one embodiment, the differentiated iPSCs are T cells prepared by a method of differentiating iPSCs into T cells. In some embodiments, the iPSCs are subjected to a differentiation protocol that includes adding recombinant human DLL-4 protein to the culture medium. In some cases, the cells are cultured in a medium containing human DLL-4 protein for the last hours of culture (e.g., 12, 18, 20, or 24 hours). In some embodiments, the recombinant human DLL-4 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the recombinant human DLL-4 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the recombinant human DLL-4 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 37, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0307] Recombinant DLL4 mutant proteins having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 37 are provided herein. In some embodiments, the recombinant DLL4 mutant protein has an amino acid sequence provided in SEQ ID NOs: 90 to 95 of US Patent Application Publication No. 2022 / 0333074, the content of which including Table 2 and the Sequence Listing is incorporated herein by reference in its entirety.

[0308] In many embodiments, the differentiated iPSCs are CD34-positive (CD34+) cells prepared by a method of differentiating iPSCs into CD34+ cells. Protocols for generating CD34+ cells from iPSCs include, for example, those disclosed in International Publication Nos. WO 2010 / 099539, WO 2010 / 141801, WO 2017 / 070333, WO 2017 / 070337, WO 2018 / 048828, WO 2019 / 157597, and WO 2020 / 252477, the content of which is incorporated herein by reference in its entirety.

[0309] In some embodiments, iPSC cells are differentiated into hematopoietic progenitor cells (HPCs). In an exemplary embodiment of the differentiation protocol, the iPSC cells are in HDM-I medium plus H1152. In some examples, the HDM medium contains IMDM medium, Ham's F12 medium, CTS B27 minus vitamin A supplement, non-essential amino acids, ascorbic acid, Mg2 phosphate, monothioglycerol, and heparin. The HDM-I medium can contain the HDM + CHIR99021 GSK3 inhibitor, FGF2, and VEGF. In some examples, the cells are further cultured in HDM-II medium containing the HDM medium in addition to BMP4, FGF2, and VEGF. In some examples, the cells are further cultured in HDM-III medium containing the HDM medium in addition to BMP4, SCF, TPO, FLT3L, and IL3. The resulting HPCs can be collected.

[0310] In some embodiments, HPCs are differentiated to generate NK or T cells. In some examples, the HPCs are cultured in a bioreactor coated with retronectin / DLL4, such as a G-Rex bioreactor. Notch signaling factors, cytokines, and growth factors can be added to the culture medium to facilitate differentiation into the lymphoid lineage and subsequent maturation and activation of NK or T cells.

[0311] In some embodiments, maturation and / or activation of NK or T cells from HPCs involves culturing the HPCs in a culture medium containing recombinant IL-12 protein. IL-12 is a cytokine that stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells. In some embodiments, the recombinant IL-12 protein comprises human IL-12 p70. In various embodiments, recombinant IL-12 comprises the human IL-12 p70 p40 subunit and the human IL-12 p70 p35 subunit. In various embodiments, the recombinant IL-12 protein comprises the human IL-12 p70 p40 subunit, the human IL-12 p70 p35 subunit, and a linker. In various embodiments, the recombinant IL-12 protein comprises, from N- to C-terminus, the human IL-12 p70 p40 subunit, a linker, and the human IL-12 p70 p35 subunit. In certain embodiments, the recombinant IL-12 protein comprises, from N- to C-terminus, the human IL-12 p70 p35 subunit, a linker, and the human IL-12 p70 p40 subunit. In some embodiments, the recombinant IL-12 protein comprises an amino acid sequence having at least 90%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 33. In various embodiments, the recombinant IL-12 protein is encoded by a polynucleotide having at least 90%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34.

[0312] Detailed descriptions for generating iPSC-derived γδ T cells are known in the art and can be found in International Publication No. WO 2022 / 216514. Briefly, using methods described in International Publication No. WO 2022 / 120334, International Publication No. WO 2022 / 216514, International Publication No. WO 2022 / 216624; International Publication No. WO 2023 / 049918, U.S. Patent No. 8,183,038, U.S. Patent No. 8,268,620, U.S. Patent No. 8,440,461, U.S. Patent No. 8,546,140, U.S. Patent No. 8,765,470, U.S. Patent No. 8,952,801, U.S. Patent No. 9,328,332, U.S. Patent No. 9,499,786, U.S. Patent No. 9,644,184, and U.S. Patent No. 10,865,381, all of which are known in the art and the entire content of each is incorporated herein by reference, peripheral blood mononuclear cells (PBMCs) from healthy donors can be reprogrammed into iPSCs. Using methods described in International Publication No. WO 2022 / 216514, which is known in the art, iPSCs can be differentiated into hematopoietic progenitor cells (HSCs) such as CD34+ HSCs. Using methods described in Examples 1 and 3 of International Publication No. WO 2022 / 216514, which is known in the art, CD34+ HSCs can be further differentiated to generate T cells, particularly γδ T cells.

[0313] Detailed descriptions for generating iPSC-derived αβ T cells are known in the art and can be found in Example 2 of International Publication No. WO 2023 / 049918, the entire content of which is incorporated herein by reference.

[0314] In some embodiments, the iPSCs and their derivatives express a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1. In some embodiments, the iPSCs and their derivatives express a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2. In some embodiments, the iPSCs and their derivatives express a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4. In some embodiments, the iPSCs and their derivatives express a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the iPSCs and their derivatives express a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the iPSCs and their derivatives express a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the iPSCs and their derivatives express a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the iPSCs and their derivatives express a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 39. In some embodiments, the iPSCs and their derivatives also express the IL-15 protein of SEQ ID NO: 16. In certain embodiments, the iPSCs and their derivatives also express the IL-15 and IL-15Rα fusion protein of SEQ ID NO: 17. In some examples, the iPSCs and their derivatives express an exogenous polynucleotide encoding the IL-15 protein of SEQ ID NO: 16. In some examples, the iPSCs and their derivatives express an exogenous polynucleotide encoding the IL-15 and IL-15Rα fusion protein of SEQ ID NO: 17. In some examples, the iPSCs and their derivatives express an exogenous polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the iPSCs and their derivatives also express the HLA-E protein of SEQ ID NO: 14. In some embodiments, the iPSCs and their derivatives also express the HLA-G protein of SEQ ID NO: 15. In some embodiments, the iPSCs and their derivatives also express an exogenous polynucleotide encoding the HLA-E protein of SEQ ID NO: 14. In some embodiments, the iPSCs and their derivatives also express an exogenous polynucleotide encoding the HLA-G protein of SEQ ID NO: 15. In some cases, the iPSCs and their derivatives express the polypeptide of SEQ ID NO: 19.In some cases, iPSCs and their derivatives express the polypeptide of SEQ ID NO: 20. In some cases, iPSCs and their derivatives express the polynucleotide of SEQ ID NO: 21 and / or SEQ ID NO: 22. In some embodiments, iPSCs and their derivatives express the chimeric antigen receptor described in International Publication No. WO 2022 / 120334, the contents of which are incorporated herein by reference in their entirety. In some embodiments, iPSCs and their derivatives express the artificial cell death polypeptide described in U.S. Patent Application Publication No. US 2022 / 0332782, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, iPSCs and their derivatives express a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, 24, 25, 27, 29, 30, or 31. In other embodiments, iPSCs and their derivatives express a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 26, 28, or 32. Examples of iPSCs and their derivatives include, but are not limited to, human iPSCs generated from reprogrammed peripheral blood mononuclear cells, human iPSCs generated by reprogrammed NK cells, human iPSCs generated by reprogrammed T cells, CD34+ hematopoietic progenitor cells derived from human iPSCs, NK cells derived from human iPSCs, T cells derived from human iPSCs, NK cells differentiated from CD34+ hematopoietic progenitor cells derived from human iPSCs, and T cells differentiated from CD34+ hematopoietic progenitor cells derived from human iPSCs. The T cells can be αβ T cells or γδ T cells.

[0315] XII. Vector In one aspect, an isolated vector (construct) is provided that includes a polynucleotide sequence encoding a useful polypeptide according to embodiments of the present disclosure. Any vector known to those skilled in the art can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressive promoter. Several expression vectors capable of delivering nucleic acids to cells are known in the art and can be used in the present invention for the production of recombinant proteins in cells. Recombinant expression vectors according to the described embodiments can be generated using conventional cloning techniques or artificial gene synthesis.

[0316] In some embodiments, any of the exogenous polynucleotides is operably linked to one or more exogenous promoters, such as, but not limited to, CAG, CMV, EF1a, PGK1, SV40, UBC, and human beta-actin, and other constitutive, inducible, time-specific, tissue-specific, and cell type-specific promoters. In some embodiments, any of the exogenous polynucleotides is operably linked to one or more endogenous promoters found in a selected genomic locus (such as a gene locus), such as, but not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, beta-2 microglobulin (B2M), GAPDH, TCR (such as TRA, TRB, TRD, TRG), and RUNX1, and other loci that meet the criteria of a genomic safe harbor.

[0317] In some embodiments, the vector comprises an exogenous polynucleotide having, in order from 5' to 3', (a) a promoter, (b) a polynucleotide sequence, and (c) a terminator / polyadenylation signal. Non-limiting examples of constitutive promoters include CAG, EF1a, UBC, CMV, SV40, PGK1, and human beta-actin. Non-limiting examples of terminator / polyadenylation signals include the SV40 signal, the BGH signal, the hGH signal, and the PGK signal.

[0318] In some embodiments, the vector comprises a left homologous arm and a right homologous arm adjacent to the exogenous polynucleotide. As used herein, "left homologous arm" and "right homologous arm" refer to a pair of nucleic acid sequences adjacent to the exogenous polynucleotide that facilitate the integration of the exogenous polynucleotide into a designated chromosomal locus. The sequences of the left and right homologous arms can be designed based on the target integration site. In some embodiments, the left or right homologous arm is homologous to the sequence on the left or right side of the integration site. In some embodiments, the left homologous arm and the right homologous arm target the CD70 gene.

[0319] XIII. Compositions In some aspects, provided are compositions or populations of iPSCs or their derivative cells that express one or more of the recombinant proteins described herein. In some embodiments, the cells are CD34+ cells, NK cells, T cells, iNK cells, or iT cells. In certain embodiments, the cells are NK cells derived from iPSCs. In some embodiments, the cells are T cells derived from iPSCs. In some examples, the T cells derived from iPSCs are αβ T cells. Under certain circumstances, the T cells derived from iPSCs are γδ T cells.

[0320] In one aspect, but not limited thereto, provided is a composition or population of iPSCs or their derivative cells that can express one or more recombinant proteins, including, but not limited to, recombinant CD16 proteins such as CD16 variant proteins, recombinant NKG2D proteins, CAR proteins, fusion proteins containing IL-15 and IL-15Ra, HLA-E, and HLA-G. In some embodiments, the composition or population of iPSCs or their derivative cells expresses recombinant CD16 proteins such as CD16 variant proteins, recombinant NKG2D proteins, and CAR proteins. In some embodiments, the composition or population of iPSCs or their derivative cells expresses recombinant CD16 proteins such as CD16 variant proteins, recombinant NKG2D proteins, CAR proteins, and a fusion protein containing IL-15 and IL-15Ra. In some embodiments, the composition or population of iPSCs or their derivative cells expresses recombinant CD16 proteins such as CD16 variant proteins, recombinant NKG2D proteins, CAR proteins, and IL-15 protein. In some embodiments, the composition or population of iPSCs or their derivative cells expresses either recombinant CD16 proteins such as CD16 variant proteins, recombinant NKG2D proteins, CAR proteins, and either HLA-E, HLA-G, or both HLA-E and HLA-G. In some embodiments, the composition or population of iPSCs or their derivative cells expresses a recombinant HSV-TK-PSMA fusion.

[0321] In some embodiments, the composition comprises a population of either NK cells, T cells, or CD34+ cells that are differentiated from iPSCs and contain an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide. In some examples, the population of NK cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide. In some embodiments, a population of either NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, and an exogenous polynucleotide construct encoding a CAR. In some embodiments, a population of either NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, and an exogenous polynucleotide construct encoding HLA-E, HLA-G, or both. In some embodiments, a population of either NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, and an exogenous polynucleotide construct encoding a fusion protein containing IL-15 and IL-15Ra. In some embodiments, a population of either NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, and an exogenous polynucleotide construct encoding an IL-15 protein.In some embodiments, a population of any of NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, an exogenous polynucleotide construct encoding a CAR, and an exogenous polynucleotide construct encoding HLA-E, HLA-G, or both. In some embodiments, a population of any of NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, an exogenous polynucleotide construct encoding a CAR, and an exogenous polynucleotide construct encoding a fusion protein containing IL-15 and IL-15Ra. In some embodiments, a population of any of NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, an exogenous polynucleotide construct encoding a CAR, and an exogenous polynucleotide construct encoding HLA-E, HLA-G, or both, and an exogenous polynucleotide construct encoding a fusion protein containing IL-15 and IL-15Ra. In some embodiments, a population of any of NK cells, T cells, or CD34+ cells differentiated from iPSCs contains an exogenous polynucleotide construct encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, an exogenous polynucleotide construct encoding a CAR, and an exogenous polynucleotide construct encoding HLA-E, HLA-G, or both, and an exogenous polynucleotide construct encoding IL-15 protein. In some embodiments, any of the cell populations can also express an exogenous polynucleotide construct containing HSV-TK-PSMA described herein.

[0322] In some examples, one or more of the exogenous polynucleotide constructs are introduced into a specific genomic locus (e.g., a gene locus). In many cases, one or more of the exogenous polynucleotide constructs are introduced into a safe harbor genomic locus (e.g., a gene locus). In some examples, one or more of the exogenous polynucleotide constructs are introduced into a gene locus such that gene expression is reduced or eliminated.

[0323] In some aspects, compositions are provided that include the isolated polynucleotides, host cells, and / or iPSCs or derivative cells thereof described herein. In certain embodiments, the composition also includes one or more therapeutic agents selected from the group consisting of peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), siRNA, oligonucleotides, mononuclear blood cells, vectors comprising one or more polynucleotides of interest, antibodies, chemotherapeutic agents, radioactive moieties or agents, or immunomodulatory agents.

[0324] In some embodiments, the composition comprises an isolated polynucleotide, host cell, and / or iPSC or a derivative cell thereof as described herein, and a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulation, or other materials well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient, or diluent depend on the route of administration. Pharmaceutically acceptable carriers include non-toxic materials that do not interfere with the effectiveness of the compositions described herein or the biological activity of the compositions described herein. The formulation of pharmaceutical active ingredients with pharmaceutically acceptable carriers is known in the art, such as Remington: The Science and Practice of Pharmacy (e.g. 21st edition (2005), and any later editions). Non-limiting examples of additional components include buffers, diluents, solvents, isotonicity regulators, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used in the formulation of the described pharmaceutical compositions.

[0325] XIV. Methods of Treating Cancer In some aspects, methods of treating a disease or disorder such as cancer and / or an autoimmune disease are provided by administering any of the cells described herein. The teachings of the present disclosure may be relevant to any and all cancers. In some embodiments, the cancer treated by the methods of the present disclosure is a solid tumor. In some embodiments, the cancer treated by the methods of the present disclosure is a hematological malignancy.

[0326] In some embodiments, NK cells described herein that are differentiated from iPSCs and engineered (e.g., modified) to express one or more of the provided exogenous polynucleotide constructs can be administered to a patient to treat cancer and / or autoimmune diseases. In some embodiments, T cells (e.g., gamma-delta T cells or γδ T cells) described herein that are differentiated from iPSCs and engineered to express one or more of the provided exogenous polynucleotide constructs can be administered to a patient to treat cancer and / or autoimmune diseases. In some embodiments, CD34+ cells described herein that are differentiated from iPSCs and engineered to express one or more of the provided exogenous polynucleotide constructs can be administered to a patient to treat cancer and / or autoimmune diseases. In many embodiments, one or more of the exogenous polynucleotide constructs include, but are not limited to, exogenous polynucleotide constructs encoding a CD16 protein (such as a high-affinity CD16 variant), an NKG2D protein, and a self-cleaving peptide, an exogenous polynucleotide construct encoding a CAR, an exogenous polynucleotide construct encoding an HLA-E protein, an exogenous polynucleotide construct encoding an HLA-G protein, an exogenous polynucleotide construct encoding an HLA-E protein and an HLA-G protein, an exogenous polynucleotide construct encoding an HLA-E protein, an HLA-G protein, and a self-cleaving peptide, an exogenous polynucleotide construct encoding a fusion protein containing IL-15 and IL-15Ra, and an exogenous polynucleotide construct encoding an IL-15 protein. In various embodiments, such cells can be administered to treat a patient having cancer, such as any type of cancer. In some embodiments, cancers treatable by the methods of the present disclosure can include glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, other solid tumor cancers, or hematological malignancies.In some embodiments, the blood malignancy is leukemia (e.g., acute lymphoblastic (ALL), chronic lymphocytic (CLL), acute myeloid (AML), chronic myeloid (CML)), myeloma, or lymphoma (e.g., Hodgkin or non-Hodgkin (NHL)).

[0327] In various embodiments of the treatment methods described herein, the disease is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, systemic lupus erythematosus, sarcoidosis, type 1 diabetes mellitus, 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-Barré syndrome, Crohn's disease, or ulcerative colitis.

[0328] In some embodiments, when administering a plurality of polypeptides having unique antigen-binding specificities via the described cells, the methods can be used to target multiple antigens (or multiple epitopes on the same antigen) in the same disease (e.g., tumor or autoimmune disease), or multiple antigens in different diseases (e.g., tumor or autoimmune disease). In various embodiments, when administering engineered cells that express a plurality of polypeptides having unique antigen-binding specificities, the described methods can be used to target multiple antigens (or multiple epitopes on the same antigen) in the same disease (e.g., tumor or autoimmune disease), or multiple antigens in different diseases (e.g., tumor or autoimmune disease).

Example

[0329] XV. Example [Example 1] Exogenous expression of CD16 in iPSC cells expressing chimeric antigen receptor (CAR) and gamma-delta iT cells derived therefrom The CD16 transgene described herein was introduced into the CD70 locus of iPSC cells carrying a CAR specific for CD19 using homologous recombination repair and CRISPR nuclease. Exemplary targeting constructs are provided in Figure 3 "p1630 CD16 of CD70" and SEQ ID NO: 13. The resulting engineered iPSC cells were differentiated into gamma / delta iT-CAR cells (iPSC-derived γδ CAR T cells) as described below.

[0330] A detailed description of useful methods for generating hematopoietic progenitor cells from iPSCs can be found in International Publication No. WO 2022 / 120334, the contents of which including examples are incorporated herein by reference in their entirety. Hematopoietic progenitor cells (HSCs expressing CD34) derived from iPSCs were differentiated into iPSC-derived γδ T cells on plates coated with RetroNectin and DLL4-Fc protein and cultured in complete medium containing basal TCDM medium supplemented with SCF (50 ng / ml), FLT3L (50 ng / ml), IL-7 (50 ng / ml), and TPO (50 ng / ml). On day 7, the cells were cultured on plates coated with RetroNectin® and DLL4-Fc protein in complete medium supplemented with CHIR09921 (final concentration of 2 μM). On days 14, 17, and 21, the cells were reseeded and cultured on plates coated with RetroNectin® and DLL4-Fc protein in complete medium supplemented with IL-2 (5 ng / ml). The culture medium was refreshed as needed. CD16 expression in the cells (see iPSC1283 and iPSC1302 in Figure 6) was evaluated from day 0 to day 21.

[0331] [Example 2] Enhanced antitumor activity of iNK cells overexpressing NKG2D protein iPSCs were engineered to constitutively express NKG2D. When the iPSCs differentiated into iNK cells, the expression of NKG2D was quantified by flow cytometry. The data demonstrated that NKG2D expression increased up to 95.5% of engineered iNK cells as compared to only 72.1% of unengineered iNK cells (Figure 7A, left).

[0332] Engineered or NKG2D-engineered iNK cells were used in a killing assay using U87 glioblastoma cells labeled with NLRs that express stress ligands (MIC-A and MIC-B) and can initiate NKG2D activity. NKG2D-engineered iNK cells killed U87 cells more potently (Figure 7A, right). To confirm that the enhanced killing was due to NKG2D expression, a neutralizing (blocking) antibody against NKG2D was used under some conditions to block the interaction between NKG2D and the stress ligand on U87 cells. When the NKG2D neutralizing antibody was included with NKG2D-engineered iNK cells, there was a marked decrease in the killing of U87 (Figure 7B). An isotype IgG1 control was used as an antibody control.

[0333] [Example 3] Enhanced antibody-dependent cellular cytotoxicity (ADCC) of iNK cells overexpressing high-affinity CD16 mutant proteins iPSCs were engineered to constitutively express one of two different naturally occurring variants of CD16 (low-affinity CD16 and high-affinity CD16). The iPSCs were then differentiated into iNK cells and used in a tumor killing assay where the target was CD20+ lymphoblastic B cells. To initiate ADCC, the anti-CD20 therapeutic antibody rituximab (black bars) was included at various concentrations. As a negative control, an unbound isotype control antibody was used under some conditions (gray bars). When iPSCs were differentiated into iNK cells expressing the low-affinity variant of CD16, ADCC was evident only when rituximab was included (an increase in dead tumor cells) (Figure 7C, upper panel). When iPSCs were differentiated into iNK cells expressing the high-affinity variant of CD16, higher ADCC was observed compared to the low-affinity version of CD16 (Figure 7C, lower panel).

[0334] [Example 4] Enhanced ADCC of CD16 overexpressed by iNK cells using a CAR-mediated tumor killing assay of fluorescently labeled Raji lymphoblastic B cells iNK cells expressing a CAR specific for CD19 (FMC63-CAR) or iNK cells without a CAR were engineered to express a CD16 variant construct - either the low-affinity CD16 variant (iPSC16) or the high-affinity CD16 variant (iPSC17 or iPSC18). In the ADCC assay, the resulting iNK cells were tested for the killing of either parental Raji or modified RajiΔCD19 target cells. Parental Raji cells are a lymphoblastic B cell line that expresses the B cell antigens CD19 and CD20. Modified RajiΔCD19 cells were modified via CRISPR gene editing to knockout the gene encoding CD19. To initiate ADCC, the anti-CD20 therapeutic antibody rituximab (bars on the right, with circles on top) was included at various concentrations. An IgG1 isotype control was included as a control (bars on the left, with squares on top).

[0335] The engineered iNK cells were co-cultured at an E:T of 3:1 with either Raji cells labeled with CellTrace Violet (CTV) or RajiΔCD19 target cells labeled with CTV in the presence of various concentrations of rituximab or host-isotype (human IgG1) control (10, 1, 0.1, 0.01, and 0 μg / mL of rituximab or host-matched isotype) for 3 hours. The percentage of CTV-positive and non-viable or dead (7-AAD-positive) cells was measured using a BD Symphony cell counter. The percentage of CTV+ / 7-AAD+ cells was graphed for each concentration of rituximab or isotype control.

[0336] ADCC was only seen in iNK cells expressing the low-affinity CD16 variant when rituximab was included. Rituximab enabled iNK cells expressing the high-affinity CD16 variant to kill Raji cells and RajiΔCD19 cells to a greater extent (Figs. 9 - 10).

[0337] Various embodiments of the invention of the present disclosure are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will be able to conceive of numerous variations, changes, and substitutions without departing from the present invention. It will be understood that in the practice of any of the inventions described herein, various alternatives to the embodiments of the invention described herein may be used.

[0338] The above detailed description has been presented to more fully illustrate some embodiments of the present invention. However, these should not be construed as limiting the broad scope of the present invention in any way. Those skilled in the art will be able to readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the present invention.

Claims

1. An induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising an exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-cleaving protease peptide, said induced pluripotent stem cell (iPSC) or a derivative cell thereof.

2. The iPSC or derivative cell thereof according to claim 1, wherein the CD16 protein is a CD16 mutant protein.

3. The iPSC or derivative cell thereof according to claim 1 or 2, wherein the CD16 mutant protein is a high-affinity CD16 mutant.

4. The iPSC or derivative cell thereof according to any one of claims 1 to 3, wherein the CD16 mutant protein is a non-cleavable CD16 mutant.

5. The iPSC or derivative cell thereof according to any one of claims 1 to 4, wherein the CD16 mutant protein comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.

6. The iPSC or derivative cell thereof according to any one of claims 1 to 5, wherein the CD16 mutant protein comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2 and 5.

7. The iPSC or derivative cell thereof according to any one of claims 1 to 6, wherein the NKG2D protein is a wild-type NKG2D protein.

8. The iPSC or derivative cell thereof according to any one of claims 1 to 6, wherein the NKG2D protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

4.

9. The iPSC or derivative cell thereof according to any one of claims 1 to 8, wherein the self-cleaving protease peptide is selected from the group consisting of Tesehovirus-1 2A (P2A) peptide, Foot-and-mouth disease virus 2A (F2A) peptide, Equine rhinitis A virus (ERAV) 2A (E2A) peptide, Thosea asigna virus 2A (T2A) peptide, Bombyx cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and Bombyx iridescent virus 2A (BmIFV2A) peptide.

10. The iPSC or a derivative cell thereof according to any one of claims 1 to 9, wherein the self-protease peptide is a P2A peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

3.

11. The iPSC or a derivative cell thereof according to any one of claims 1 to 10, wherein the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

6.

12. The iPSC or a derivative cell thereof according to any one of claims 1 to 11, wherein the exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPBP locus, NLRRC5 locus, RFXANK locus, RFX5 locus, RFXAP locus, CISH locus, CBLB locus, SOCS2 locus, PD1 locus, CTLA4 locus, LAG3 locus, TIM3 locus, and TIGIT locus.

13. The iPSC or a derivative cell thereof according to any one of claims 1 to 12, wherein the exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, TAP1 locus, TAP2 locus, TAPBP locus, TRAC locus, TRBC1 locus, RFXANK locus, RFX5 locus, and RFXAP locus, whereby the expression of the gene is disrupted.

14. The iPSC or a derivative cell thereof according to any one of claims 1 to 13, wherein the exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CD70 locus, CLYBL locus, NKG2A locus, and TRAC locus, whereby the expression of the gene is disrupted.

15. The iPSC or a derivative cell thereof according to claim 13 or 14, wherein the disruption of the gene comprises elimination or reduction of the expression of the gene.

16. The iPSC or a derivative cell thereof according to any one of claims 12 to 14, wherein the integration into the gene locus is caused by targeted genome editing.

17. The iPSC or a derivative cell thereof according to claim 16, wherein the targeted genome editing uses a method selected from the group consisting of a CRISPR method, a zinc finger nuclease method, a TALEN method, a homing nuclease method, a homologous recombination method, and any functional modification thereof.

18. The iPSC or a derivative cell thereof according to any one of claims 1 to 17, further comprising disruption of one or more genes selected from the group consisting of the AAVS1 gene, the B2M gene, the CIITA gene, the CD70 gene, the CLYBL gene, the NKG2A gene, the NKG2D gene, the TAP1 gene, the TAP2 gene, the TAPBP gene, the TRAC gene, the TRBC1 gene, the RFXANK gene, the RFX5 gene, the RFXAP gene, and any combination thereof.

19. The iPSC or a derivative cell thereof according to claim 18, wherein the disruption is disruption of the B2M gene and the CIITA gene.

20. The iPSC or a derivative cell thereof according to claim 18 or 19, wherein the disruption of the one or more genes includes elimination or reduction of expression of the one or more genes.

21. The iPSC or a derivative cell thereof according to any one of claims 18 to 20, wherein the disruption of the one or more genes is caused by targeted genome editing.

22. The iPSC or a derivative cell thereof according to claim 21, wherein the targeted genome editing uses a method selected from the group consisting of a CRISPR method, a zinc finger nuclease method, a TALEN method, a homing nuclease method, a homologous recombination method, and any functional modification thereof.

23. The iPSC or a derivative cell thereof according to any one of claims 1 to 22, further comprising a second exogenous polynucleotide encoding an IL-15 protein.

24. The iPSC or a derivative cell thereof according to claim 23, wherein the IL-15 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

16.

25. An iPSC or a derivative cell thereof according to any one of claims 1 to 22, further comprising a second exogenous polynucleotide encoding a fusion polypeptide comprising IL-15 and interleukin-15 receptor alpha (IL-15Rα).

26. The iPSC or a derivative cell thereof according to claim 25, wherein the fusion polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

17.

27. The iPSC or a derivative cell thereof according to claim 25 or 26, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

17.

28. An iPSC or a derivative cell thereof according to any one of claims 1 to 27, further comprising a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) protein.

29. The iPSC or a derivative cell thereof according to claim 27, wherein the HLA-E comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

14.

30. An iPSC or a derivative cell thereof according to any one of claims 1 to 29, further comprising a fourth exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G) protein.

31. The iPSC or a derivative cell thereof according to claim 30, wherein the HLA-G comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

15.

32. The iPSC or a derivative cell thereof according to claim 30 or 31, wherein the HLA-E protein and the HLA-G protein are operably linked by a second self-cleaving peptide.

33. The iPSC or a derivative cell thereof according to claim 32, wherein the second self-cleaving peptide is selected from the group consisting of a P2A peptide, an F2A peptide, an E2A peptide, a T2A peptide, a BmCPV2A peptide, and a BmIFV2A peptide.

34. The iPSC or its derivative cell according to any one of claims 23 to 33, wherein the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, B2M locus, CII TA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPBP locus, NLR C5 locus, RFXANK locus, RFX5 locus, RFXAP locus, CISH locus, CBLB locus, SOCS2 locus, PD1 locus, CTLA4 locus, LAG3 locus, TIM3 locus, and TIGIT locus, and any combination thereof.

35. The iPSC or its derivative cell according to any one of claims 23 to 34, wherein the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, B2M locus, CII TA locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, TAP1 locus, TAP2 locus, TAPBP locus, TRAC locus, TRBC1 locus, RFXANK locus, RFX5 locus, RFXAP locus, and any combination thereof.

36. The iPSC or its derivative cell according to any one of claims 23 to 35, wherein the second, third, and / or fourth exogenous polynucleotide is integrated within one or more gene loci selected from the group consisting of the AAVS1 locus, B2M locus, CII TA locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, TAP1 locus, TAP2 locus, TAPBP locus, TRAC locus, TRBC1 locus, RFXANK locus, RFX5 locus, RFXAP locus, and any combination thereof, whereby the one or more genes are disrupted.

37. The iPSC or a derivative cell thereof according to any one of claims 23 to 36, wherein the second, third, and / or fourth exogenous polynucleotide is integrated into one or more gene loci selected from the group consisting of the AAVS1 locus, B2M locus, CII TA locus, CD70 locus, CLYBL locus, NKG2A locus, TRAC locus, and any combination thereof, whereby the one or more genes are disrupted.

38. The iPSC or a derivative cell thereof according to claim 36 or 37, wherein the disruption in the one or more genes comprises elimination or reduction of expression of the one or more genes.

39. The iPSC or a derivative cell thereof according to any one of claims 1 to 38, wherein the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs).

40. The iPSC or a derivative cell thereof according to any one of claims 1 to 39, wherein the iPSC is derived from reprogrammed NK or T cells.

41. The iPSC or a derivative cell thereof according to any one of claims 1 to 40, further comprising a fifth exogenous polynucleotide encoding a chimeric antigen receptor (CAR) that binds to a target antigen.

42. The iPSC or its derivative cells according to claim 41, wherein the target antigen is selected from the group consisting of 17-1A antigen, A3, A33 antigen, AFP, B7H4, Ba733, BCMA, BrE3 antigen, CA125, CA9 (CAIX), CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD22, CD23, CD25, CD30, CD33, CD33, CD38, CD45, CD70, CD74, CD79, CD79a, CD80, CD123, CD133, CD138, CEACAM5, CEACAM6, CLDN18.2, CLL1, cMET, colon-specific antigen-p (CSAp), ED-B fibronectin, EGFR, EGFRvIII, EGP-1, EGP-2, EpCAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FGFR1, FGFR3, Flt-1, Flt-3, FOLR1, FOLR2, FOLR3, FSHR, GD2, GPC-3, GPRC5D, HCG, HCG subunit, HER2, HIF-I, HLA-DR, Ia, IGF-I, IL13Rα2, IL-2, IL-6, IL-8, KC4 antigen, KS-1 antigen, KS1-4 antigen, Le-Y, MAGE, MET, MIF, MSLN, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA90, NCA95, nectin-4, p53, PAP, PDGFRA, PLGF, PSA, PSMA, ROBO1, RS5, S100, SLAM F7, SLITRK6, TAC, TAG-72, tenascin-C, tenascin-R, tenascin-W, tenascin-X, Thomson-Friedenreich antigen, Tn antigen, TRAILR1, TRAILR2, TRAILR3, TRAILR4, VEGF, tumor necrosis antigen, angiogenesis antigen, and oncogene antigen.

43. The iPSC or its derivative cells according to claim 41 or 42, wherein the CAR comprises an antigen-binding domain selected from the group consisting of any of those provided in Tables 1, 2, and 3.

44. The CAR is (i) a signal peptide, and (ii) an extracellular domain comprising a binding domain that specifically binds to the target antigen, (iii) a hinge region, and (iv) a transmembrane domain (v) an intracellular signaling domain, and (vi) one or more co-stimulatory domains An iPSC or a derivative cell thereof according to any one of claims 41 to 43, comprising:

45. The iPSC or a derivative cell thereof according to claim 44, wherein the signal peptide comprises a GMCSFR signal peptide.

46. The iPSC or a derivative cell thereof according to claim 44 or 45, wherein the extracellular domain comprises a single-chain Fv (scFv) or VHH domain that specifically binds to the target antigen.

47. The iPSC or a derivative cell thereof according to any one of claims 44 to 46, wherein the hinge region comprises a CD28 hinge region.

48. The iPSC or a derivative cell thereof according to any one of claims 44 to 47, wherein the transmembrane domain comprises a CD28 transmembrane domain.

49. The iPSC or a derivative cell thereof according to any one of claims 44 to 48, wherein the intracellular signaling domain comprises a CD3ζ intracellular domain.

50. The iPSC or a derivative cell thereof according to any one of claims 44 to 49, wherein the one or more co-stimulatory domains comprise a CD28 signaling domain.

51. A derivative cell thereof according to any one of claims 1 to 50, which is an NK cell or a T cell.

52. A derivative cell thereof according to claim 51, which is an NK cell.

53. A derivative cell thereof according to claim 51, which is a T cell.

54. A derivative cell thereof according to any one of claims 1 to 53, which is a CD34+ hematopoietic progenitor cell.

55. A composition comprising a population of iPSCs or derivative cells thereof according to any one of claims 1 to 54.

56. (i) a first exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-protease peptide, the first exogenous polynucleotide; (ii) a second exogenous polynucleotide encoding an exogenous polypeptide comprising an IL-15 protein; (iii) Optionally, a third exogenous polynucleotide encoding human leukocyte antigen E (HLA-E) and / or a fourth exogenous polynucleotide encoding human leukocyte antigen G (HLA-G) An engineered cell comprising:

57. A fifth polynucleotide encoding a combinatorial artificial cell death / reporter system polypeptide comprising an intracellular domain having herpes simplex virus thymidine kinase (HSV-TK) and a linker, a transmembrane region, and an extracellular domain comprising a prostate-specific membrane antigen (PSMA) extracellular domain or a fragment thereof, further comprising the engineered cell according to claim 56.

58. The engineered cell according to claim 56 or 57, wherein the HSV-TK comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23 or 29.

59. The engineered cell according to any one of claims 56 to 58, wherein the combinatorial artificial cell death / reporter system polypeptide comprises HSV-TK fused to a mutant PSMA polypeptide cleaved via the linker.

60. The engineered cell according to any one of claims 56 to 59, wherein the cleaved mutant PSMA polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

24.

61. The engineered cell according to any one of claims 56 to 59, wherein the linker comprises a self-protease peptide sequence selected from the group consisting of a P2A peptide sequence, a T2A peptide sequence, an E2A peptide sequence, and an F2A peptide sequence.

62. The engineered cell according to any one of claims 56 to 60, wherein the artificial cell death / reporter system polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:

25.

63. The engineered cell according to any one of claims 56 to 62, wherein the artificial cell death / reporter system polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 27, 30, and 31.

64. The engineered cell according to any one of claims 56 to 63, wherein the artificial cell death / reporter system polypeptide comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 26, 28, and 32.

65. (i)a first exogenous polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-protease peptide, the first exogenous polynucleotide; (ii)a second exogenous polynucleotide encoding a fusion polypeptide comprising an IL-15 protein and an IL-15 receptor alpha (IL-15Rα) protein; (iii)optionally, a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) protein and / or a fourth exogenous polynucleotide encoding a human leukocyte antigen G (HLA-G) protein An engineered cell comprising the same.

66. The engineered cell according to any one of claims 56 to 65, which is an engineered induced pluripotent stem cell (iPSC), an engineered natural killer (NK) cell, or an engineered T cell.

67. The engineered cell according to any one of claims 56 to 66, wherein the first exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

6.

68. The engineered cell according to any one of claims 56 to 67, wherein the IL-15 protein comprises the amino acid sequence of SEQ ID NO:

16.

69. The engineered cell according to any one of claims 56 to 67, wherein the second exogenous polynucleotide comprises a nucleic acid sequence encoding an IL-15 / IL-15Rα fusion protein of SEQ ID NO:

17.

70. The engineered cell according to any one of claims 56 to 69, wherein the third exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 21 and the fourth exogenous polynucleotide comprises the nucleic acid sequence of SEQ ID NO:

22.

71. The engineered cell according to any one of claims 56 to 69, wherein the HLA-E protein and the HLA-G protein are linked by a self-protease peptide.

72. The engineered cell according to any one of claims 56 to 70, further comprising disruption of the B2M and CIITA genes.

73. The engineered cell according to claim 72, wherein the disruption of the B2M and CIITA genes is caused by targeted genome editing.

74. The engineered cell according to claim 73, wherein the targeted genome editing comprises using a method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variant thereof.

75. The engineered cell according to any one of claims 56 to 74, wherein the first exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.

76. The engineered cell according to any one of claims 56 to 75, wherein the second exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, the B2M locus, the CIITA locus, the CCR5 locus, the CD70 locus, the CLYBL locus, the NKG2A locus, the NKG2D locus, the CD38 locus, the TRAC locus, the TRBC1 locus, the ROSA26 locus, the collagen locus, the HTRP locus, the GAPDH locus, the RUNX1 locus, the TAP1 locus, the TAP2 locus, the TAPBP locus, the NLRRC5 locus, the RFXANK locus, the RFX5 locus, the RFXAP locus, the CISH locus, the CBLB locus, the SOCS2 locus, the PD1 locus, the CTLA4 locus, the LAG3 locus, the TIM3 locus, and the TIGIT locus.

77. The manipulated cell according to any one of claims 56 to 76, wherein the third exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, collagen locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPBP locus, NLRRC5 locus, RFXANK locus, RFX5 locus, RFXAP locus, CISH locus, CBLB locus, SOCS2 locus, PD1 locus, CTLA4 locus, LAG3 locus, TIM3 locus, and TIGIT locus.

78. The manipulated cell according to any one of claims 56 to 77, wherein the fourth exogenous polynucleotide is integrated within a gene locus selected from the group consisting of the AAVS1 locus, B2M locus, CIITA locus, CCR5 locus, CD70 locus, CLYBL locus, NKG2A locus, NKG2D locus, CD38 locus, TRAC locus, TRBC1 locus, ROSA26 locus, collagen locus, HTRP locus, GAPDH locus, RUNX1 locus, TAP1 locus, TAP2 locus, TAPBP locus, NLRRC5 locus, RFXANK locus, RFX5 locus, RFXAP locus, CISH locus, CBLB locus, SOCS2 locus, PD1 locus, CTLA4 locus, LAG3 locus, TIM3 locus, and TIGIT locus.

79. The manipulated cell according to any one of claims 56 to 78, wherein the first exogenous polynucleotide and the second, third, or fourth exogenous polynucleotide are integrated within the B2M gene locus and the CIITA gene locus, thereby disrupting the B2M and CIITA genes.

80. The manipulated cell according to any one of claims 56 to 78, wherein the first exogenous polynucleotide is integrated within the CD70 locus and the second exogenous polynucleotide is integrated within the B2M gene locus, thereby disrupting the CD70 and B2M genes.

81. The first exogenous polynucleotide is integrated into the CD70 locus, and the second exogenous polynucleotide is integrated into the CIITA gene locus, whereby the CD70 and CIITA genes are disrupted. The engineered cell according to any one of claims 56 to 78.

82. The first exogenous polynucleotide is integrated into the CD70 locus, and the third or fourth exogenous polynucleotide is integrated into the B2M gene locus, whereby the CD70 and B2M genes are disrupted. The engineered cell according to any one of claims 56 to 78.

83. The first exogenous polynucleotide is integrated into the CD70 locus, and the third or fourth exogenous polynucleotide is integrated into the CIITA gene locus, whereby the CD70 and CIITA genes are disrupted. The engineered cell according to any one of claims 56 to 78.

84. The integration into the CD70 locus is into exon 1 of the CD70 gene. The engineered cell according to any one of claims 80 to 83.

85. The integration into the gene locus is caused by targeted genome editing. The engineered cell according to any one of claims 75 to 84.

86. The targeted genome editing includes using a method selected from the group consisting of the CRISPR method, the zinc finger nuclease method, the TALEN method, the homing nuclease method, the homologous recombination method, and any functional variant thereof. The engineered cell according to claim 85.

87. The engineered cell according to any one of claims 78 to 86, further comprising a fifth exogenous polynucleotide encoding a chimeric antigen receptor (CAR) that binds to a target antigen.

88. The engineered cell according to claim 87, wherein the target antigen is selected from the group consisting of 17-1A antigen, A3, A33 antigen, AFP, B7H4, Ba733, BCMA, BrE3 antigen, CA125, CA9 (CAIX), CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD22, CD23, CD25, CD30, CD33, CD33, CD38, CD45, CD70, CD74, CD79, CD79a, CD80, CD123, CD133, CD138, CEACAM5, CEACAM6, CLDN18.2, CLL1, cMET, colon-specific antigen-p (CSAp), ED-B fibronectin, EGFR, EGFRvIII, EGP-1, EGP-2, EpCAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FGFR1, FGFR3, Flt-1, Flt-3, FOLR1, FOLR2, FOLR3, FSHR, GD2, GPC-3, GPRC5D, HCG, HCG subunit, HER2, HIF-I, HLA-DR, Ia, IGF-I, IL13Rα2, IL-2, IL-6, IL-8, KC4 antigen, KS-1 antigen, KS1-4 antigen, Le-Y, MAGE, MET, MIF, MSLN, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA90, NCA95, nectin-4, p53, PAP, PDGFRA, PLGF, PSA, PSMA, ROBO1, RS5, S100, SLAM F7, SLITRK6, TAC, TAG-72, tenascin-C, tenascin-R, tenascin-W, tenascin-X, Thomson-Friedenreich antigen, Tn antigen, TRAILR1, TRAILR2, TRAILR3, TRAILR4, VEGF, tumor necrosis antigen, angiogenesis antigen, and oncogene antigen.

89. The engineered cell according to claim 88, wherein the CAR comprises an antigen-binding domain selected from the group consisting of any of those provided in Tables 1, 2, and 3.

90. The CAR is (i) a signal peptide, (ii) an extracellular domain comprising a binding domain that specifically binds to the target antigen, (iii) a hinge region, (iv) a transmembrane domain, and (v) an intracellular signaling domain. (vi) one or more costimulatory domains and an engineered cell according to any one of claims 87 to 89. **Claim 91** The engineered cell according to claim 90, wherein the signal peptide comprises a GMCSFR signal peptide. **Claim 92** The engineered cell according to claim 90 or 91, wherein the extracellular domain comprises a single-chain Fv (scFv) or VHH domain that specifically binds to the target antigen. **Claim 93** The engineered cell according to any one of claims 90 to 92, wherein the hinge region comprises a CD28 hinge region. **Claim 94** The engineered cell according to any one of claims 90 to 93, wherein the transmembrane domain comprises a CD28 transmembrane domain. **Claim 95** The engineered cell according to any one of claims 90 to 94, wherein the intracellular signaling domain comprises a CD3ζ intracellular domain. **Claim 96** The engineered cell according to any one of claims 90 to 95, wherein the one or more costimulatory domains comprise a CD28 signaling domain. **Claim 97** The engineered cell according to any one of claims 56 to 96, wherein the engineered iPSC has been differentiated into an engineered differentiated cell. **Claim 98** The engineered cell according to any one of claims 56 to 97, wherein the engineered iPSC has been differentiated into an engineered NK cell. **Claim 99** The engineered cell according to any one of claims 56 to 97, wherein the engineered iPSC has been differentiated into an engineered T cell. **Claim 100** The engineered cell according to any one of claims 56 to 97, wherein the engineered iPSC has been differentiated into an engineered CD34+ hematopoietic progenitor cell. **Claim 101** A composition comprising a population of engineered iPSCs according to any one of claims 56 to 96. **Claim 102** A composition comprising a population of engineered differentiated cells according to any one of claims 56 to 101. **Claim 103** A composition comprising a population of engineered NK cells according to any one of claims 56 to 98. **Claim 104** A composition comprising a population of engineered T cells according to any one of claims 56 to 97 and 99. **Claim 105** A composition comprising a population of engineered CD34+ hematopoietic progenitor cells according to any one of claims 56 to 97 and 100. **Claim 106** A method of treating cancer in a subject in need thereof, comprising administering to the subject in need thereof a derivative cell according to any one of claims 1 to 54, an engineered NK cell according to any one of claims 56 to 98, an engineered T cell according to any one of claims 56 to 97 and 99, an engineered CD34+ hematopoietic progenitor cell according to any one of claims 56 to 97 and 100, or a composition according to any one of claims 55 and 101 to 105.

107. The method according to claim 106, wherein the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoma, benign lesion, bladder cancer, bone cancer, breast cancer, thyroid cancer, laryngeal carcinoma, lung carcinoma, oral carcinoma, pharyngeal carcinoma, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), cutaneous melanoma, endocrine cancer, endometrial cancer, gastrointestinal cancer, genitourinary cancer, glioblastoma, head and neck cancer, hematological malignancy, hematopoietic cancer, Hodgkin lymphoma, intraocular melanoma, leukemia, liver cancer, lymphoma, melanoma, myeloma, myeloproliferative disorder, nervous system cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, papilloma, parathyroid cancer, prostate cancer, renal cell cancer, sarcoma, skin cancer, solid tissue carcinoma, squamous cell carcinoma, and uterine cancer.

108. A method of differentiating the iPSC cells into NK cells, comprising subjecting the iPSC cells according to any one of claims 1 to 50 and 56 to 96 to a differentiation protocol, thereby generating the NK cells, wherein the differentiation protocol comprises culturing the cells in a medium containing recombinant human IL-12 protein during the last 24 hours of culture under the differentiation protocol.

109. The method according to claim 108, wherein the recombinant human IL-12 protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

33.

110. A method for differentiating the iPSC cells into T cells, comprising subjecting the iPSC cells according to any one of claims 1 to 50 and 56 to 96 to a differentiation protocol including culturing the cells in a medium containing a recombinant DLL4 variant polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 37, thereby producing the T cells.

111. A recombinant DLL4 variant polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 35 to 37.

112. A method for differentiating the iPSC cells into CD34+ hematopoietic progenitor cells, comprising subjecting the iPSC cells according to any one of claims 1 to 50 and 56 to 96 to a differentiation protocol including culturing the cells in a preselected medium, thereby producing the CD34+ hematopoietic progenitor cells.

113. A polynucleotide encoding a CD16 protein and an NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by a self-protease peptide.

114. The polynucleotide according to claim 113, wherein the CD16 protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

5.

115. The polynucleotide according to claim 113 or 114, wherein the CD16 protein is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

7.

116. The polynucleotide according to claim 113, wherein the CD16 protein is a CD16 variant protein.

117. The polynucleotide according to claim 115, wherein the CD16 variant protein comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.

118. The polynucleotide according to claim 115 or 117, wherein the CD16 variant comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or SEQ ID NO:

5.

119. The polynucleotide according to any one of claims 115 to 118, wherein the CD16 variant is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

7.

120. The polynucleotide according to any one of claims 115 to 119, wherein the NKG2D protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

4.

121. The polynucleotide according to any one of claims 115 to 120, wherein the NKG2D protein is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

9.

122. The polynucleotide according to any one of claims 115 to 120, wherein the NKG2D protein is an NKG2D variant protein.

123. The polynucleotide according to claim 121, wherein the NKG2D variant comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

4.

124. The polynucleotide according to any one of claims 113 to 123, wherein the self-cleaving protease peptide is selected from the group consisting of the tesehovirus-1 2A (P2A) peptide, the foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, the equine rhinitis A virus (ERAV) 2A (E2A) peptide, the zosea asignavirus 2A (T2A) peptide, the cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and the flacherie virus 2A (BmIFV2A) peptide.

125. The polynucleotide according to any one of claims 113 to 124, wherein the self-cleaving protease peptide is a P2A peptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

3.

126. The polynucleotide according to any one of claims 113 to 125, wherein the self-cleaving protease peptide is a P2A peptide encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

127. The polynucleotide according to any one of claims 113 to 125, wherein the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

6.

128. The polynucleotide according to any one of claims 113 to 126, wherein the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein has the nucleic acid sequence of SEQ ID NO:

6.

129. A vector comprising the polynucleotide according to any one of claims 113 to 127.

130. From 5' to 3', (i) a left homologous sequence, (ii) a promoter, (iii) the polynucleotide according to any one of claims 113 to 127, (iv) a terminator and / or a polyadenylation signal sequence, (iv) a right homologous sequence The vector according to claim 128, comprising.

131. The vector according to claim 130, wherein the left homologous sequence comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

11.

132. The vector according to claim 130 or 131, wherein the right homologous sequence comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

12.

133. The vector according to any one of claims 128 to 132, comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

13.

134. The vector according to any one of claims 128 to 132, comprising the nucleic acid sequence of SEQ ID NO:

13.

135. The vector according to any one of claims 128 to 132, comprising a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

39.

136. The vector according to any one of claims 128 to 132, comprising the nucleic acid sequence of SEQ ID NO: 39.

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